Anti-human VEGF receptor Flt-1 monoclonal antibody

ABSTRACT

The present invention provides an antibody or peptide which immunologically reacts with human VEGF receptor Flt-1 and cells in which human VEGF receptor Flt-1 is expressed on the cell surface and an antibody or peptide which inhibits binding of human VEGF to human VEGF receptor Flt-1. It also provides a means for the diagnosis or treatment of diseases in which their morbid states progress by abnormal angiogenesis, such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, psoriasis, and the like.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. application Ser. No.09/453,718, (allowed), filed Dec. 3, 1999 now U.S. Pat. No. 6,986,890,which is a continuation-in-part of U.S. Ser. No. 09/315,051, filed May20, 1999 now abandoned, which is a continuation-in-part of U.S. Ser. No.09/119,014, filed Jul. 20, 1998 now U.S. Pat. No. 6,617,160, which is acontinuation-in-part of PCT/JP97/04259, filed Nov. 21, 1997, whichclaims benefit of Japan Hei. 8-311109, filed Nov. 21, 1996, Japan Hei.10-139000, filed May 20, 1998, PCT/JP97/04259, filed Nov. 21, 1997 andPCT/JP99/02661, filed May 20, 1999, the entire contents of each of whichis hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an antibody or a peptide capable ofspecifically binding to human VEGF receptor Flt-1 which is useful forthe diagnosis or treatment of diseases in which their morbid statesprogress by abnormal angiogenesis, such as proliferation or metastasisof solid tumors, arthritis in rheumatoid arthritis, diabeticretinopathy, retinopathy of prematurity and psoriasis; a hybridomacapable of producing the antibody; a method for immunologicallydetecting human VEGF receptor Flt-1 using the antibody or the peptide;and a diagnostic method and a therapeutic method for diseases, such assolid tumor, rheumatoid arthritis, diabetic retinopathy, retinopathy ofprematurity, psoriasis and the like, using the antibody or the peptide.

2. Brief Description of the Background Art

Angiogenesis plays an important role in the individual development andconstruction of tissues in vertebrates, is directly involved in theformation of the corpus luteum during the sexual cycle, transientproliferation of the uterine endometrium and formation of the placentain mature individuals (females). With regard to pathological states,angiogenesis is involved in the proliferation or metastasis of solidtumors and formation or acceleration of morbidity in diabeticretinopathy and rheumatoid arthritis [J. Biol. Chem., 267: 10931(1992)]. Angiogenesis occurs by the secretion of an angiogenesis factorand involves the process of a tube formation and producing a new bloodvessel. During this process, the basement membrane and interstitum aredestroyed by a protease secreted from endothelial cells of an existingblood vessel around the secreted angiogenesis factor, followed bysubsequent migration and proliferation of vascular endothelial cells [J.Biol. Chem., 267: 10931 (1992)]. Factors which induce angiogenesisinclude vascular permeability factor (hereinafter “VPF”) and vascularendothelial growth factor (hereinafter “VEGF”) (hereinafter “VPF/VEGF”).These factors are considered the most important factors in pathologicaland non-pathological angiogenesis [Advances in Cancer Research, 67: 281(1995)]. VPF/VEGF is a protein having a molecular weight of about 40,000constituted by homodimers, which had been reported to be independentmolecules as vascular permeability factor (VPF) in 1983 [Science, 219:983 (1983)] and as vascular endothelial growth factor (VEGF) in 1989[Biochem. Biophys. Res. Comm., 161: 851 (1989)], but it has beenrevealed as the results of cDNA cloning that they are the same substance[Science, 246: 1306 (1989); Science, 246: 1309 (1989)] (hereinafter, theterm “VPF/VEGF” is recited as “VEGF”). Beyond the activity of VEGF uponvascular endothelial cells described above, VEGF has also been shown tohave a growth enhancing activity [Biochem. Biophys. Res. Comm., 161: 851(1989)], a migration enhancing activity [J. Immunology, 152: 4149(1994)], a metalloprotease secretion enhancing activity [J. CellPhysiol., 153: 557 (1992)], a urokinase and tPA secretion enhancingactivity [Biochem. Biophys. Res. Comm., 181: 902 (1991)], and the like.Furthermore, VEGF has been shown to have an angiogenesis enhancingactivity [Circulation, 92 suppl II: 365 (1995)], a vascular permeabilityenhancing activity [Science, 219: 983 (1983)], and the like as its invivo activities. It has been reported that VEGF is a growth factorhaving extremely high specificity for vascular endothelial cells[Biochem. Biophys. Res. Comm., 161: 851 (1989)] and that four proteinshaving different molecular weight are present due to alternativesplicing of mRNA [J. Biol. Chem., 267: 26031 (1991)].

Among diseases accompanied by angiogenesis, it has been reported thatVEGF plays an important role in the proliferation or metastasis of solidtumors and formation of morbid states of diabetic retinopathy andrheumatoid arthritis. With regard to solid tumors, production of VEGF ina number of human tumor tissues has been reported, such as in renalcarcinoma [Cancer Research, 54: 4233 (1994)], breast cancer [HumanPathology, 26: 86 (1995)], brain tumor [J. Clinical Investigation, 91:153 (1993)], gastrointestinal cancer [Cancer Research, 53: 4727 (1993)],ovarian cancer [Cancer Research, 54: 276 (1994)], and the like. Also,results of a study on the correlation between VEGF expression quantityin tumors and survival ratio of patients in patients with breast cancerhave revealed that tumor angiogenesis is more active in tumorsexpressing high levels of VEGF than low VEGF expression tumors and thatthe survival ratio is lower in breast cancer patients having high VEGFexpression tumors than breast cancer patients having low VEGF expressiontumors [Japanese J. Cancer Research, 85: 1045 (1994)]. It has beenreported also that an anti-VEGF monoclonal antibody inhibited tumorgrowth in a xenograft model test system in which a human tumor wastransferred into nude mice by subcutaneous transplantation [Nature, 362:841 (1993)]. Also, it has been reported that, in a metastatic cancermodel of a human tumor in nude mice, an anti-VEGF monoclonal antibodyinhibited metastasis of the tumor [Cancer Research, 56: 921 (1996)].Additionally, since a high concentration of VEGF was detected in humancarcinomatous pleural perfusions and ascites, the possibility that VEGFis a major factor involved in the retention of pleural perfusions andascites has been suggested [Biochimica et Biophysica Acta, 1221: 211(1994)].

In diabetic retinopathy, abnormal angiogenesis causes retinal detachmentand hemorrhage of the vitreous body, resulting in blindness, and it hasbeen reported that angiogenesis in diabetic retinopathy and theexpression level of VEGF in the patient's eye balls are positivelycorrelative [New England J. Medicine, 331: 1480 (1994)]. Also, it hasbeen reported that angiogenesis in a monkey retinopathy model isinhibited when the VEGF activity is inhibited by the intraocularadministration of an anti-VEGF neutralizing monoclonal antibody [Arch.Ophthalmol., 114: 66 (1996)].

Progress in the morbid states of rheumatoid arthritis (destruction ofbone and cartilage) is accompanied by angiogenesis, and it has beenreported that a high concentration of VEGF is contained in the synovialfluid of patients with rheumatoid arthritis and that macrophages injoints of patients with rheumatoid produce VEGF rheumatoid arthritis[Journal of Immunology, 152: 4149 (1994); J. Experimental Medicine, 180:341 (1994)].

VEGF receptors have been reported. These include fms-like tyrosinekinase (referred to as “Flt-1” hereinafter) [Oncogene, 5: 519 (1990);Science, 255: 989 (1992)] and kinase insert domain-containing receptor(referred to as “KDR” hereinafter) [WO 92/14748; Proc. Natl. Acad. Sci.USA, 88: 9026 (1991)]; Biochem. Biophys. Res. Comm., 187: 1579 (1992);WO 94/11499); which belong to the receptor type tyrosine kinase family.Each of Flt-1 and KDR is a membrane protein of 180 to 200 kilodalton inmolecular weight which has an extracellular domain consisting of 7immunoglobulin-like regions and an intracellular domain consisting of atyrosine kinase region. It has been reported that VEGF specificallybinds to Flt-1 and KDR at Kd values of 20 pM and 75 pM and that Flt-1and KDR are expressed in vascular endothelial cells in a specific manner[Proc. Natl. Acad. Sci. USA, 90: 7533 (1993); Proc. Natl. Acad. Sci.USA, 90: 8915 (1993)]. With regard to Flt-1 in various diseases, it hasbeen reported that, in comparison with vascular endothelial cells innormal tissues, expression of Flt-1 mRNA increases in tumor vascularendothelial cells of human glioblastoma tissues [Nature, 359: 845(1992)] and tumor vascular endothelial cells of human digestive organcancer tissues [Cancer Research, 53: 4727 (1993)]. Additionally, it hasbeen reported that expression of Flt-1 mRNA is observed by in situhybridization in vascular endothelial cells of joints of patients withrheumatoid arthritis [J. Experimental Medicine, 180: 341 (1994)]. Theseresults strongly suggest that a VEGF/VEGF receptor Flt-1 system plays animportant role in tumor angiogenesis. Although it has been reported thatVEGF binds to Flt-1 and the intracellular domain is auto-phosphorylated[Science, 255: 989 (1992)], the detailed function of the receptormechanism is still unclear. However, it has been discovered that knockout mice in which the Flt-1 gene was destroyed die after a fetal age of8.5 to 9.5 days due to abnormal blood vessel construction caused byabnormal morphology of vascular endothelial cells during blood islandformation in the early stage of development and subsequent angiogenesis.This had led to an assumption that Flt-1 has a function essential forthe tube formation of vascular endothelial cells in angiogenesis[Nature, 376: 66 (1995)].

In view of the above, it is expected that an antibody which can inhibitbiological activities of VEGF through its binding to VEGF receptor Flt-1will be useful for the diagnosis or treatment of diseases in which theirmorbid states progress by abnormal angiogenesis, such as proliferationor metastasis of solid tumors, arthritis in rheumatoid arthritis,diabetic retinopathy, retinopathy of prematurity and psoriasis. However,an anti-VEGF receptor Flt-1 monoclonal antibody which can detect cellsin which VEGF receptor Flt-1 is expressed and anti-VEGF receptor Flt-1monoclonal antibody which can inhibit biological activities of VEGF hasnot been described in the art.

Generally, when a monoclonal antibody derived from non-human animal isadministered to human, the monoclonal antibody is recognized as aforeign material so that an antibody against the monoclonal antibodyderived from non-human animal is produced in the body. As a result, theantibody is allowed to react with the monoclonal antibody derived fromnon-human animal, and it is known that side effects are caused [J. Clin.Oncol., 2: 881 (1984); Blood, 65: 1349 (1985); J. Natl. Cancer Inst.,80: 932 (1988); Proc. Natl. Acad. Sci. USA, 82: 1242 (1985)], themonoclonal antibody is shortly cleared [J. Nucl. Med., 26: 1011 (1985);Blood, 65: 1349 (1985); J. Natl. Cancer Inst., 80: 937 (1988)], and thatthe treating effect of the antibody is decreased [J. Immunol., 135: 1530(1985); Cancer Res., 46: 6489 (1986)].

In order to solve the above problems, it has been attempted that amonoclonal antibody derived from non-human animal is modified to ahumanized antibody, such as a human chimeric antibody or a humanCDR-grafted antibody (reconstructed human antibody), using geneengineering technique. The human chimeric antibody is an antibody inwhich an antibody variable region (V region) is derived from a non-humananimal antibody and an antibody constant region (C region) is derivedfrom a human antibody [Proc. Natl. Acad. Sci. USA, 81: 6851 (1984)]. Itis reported that when the human chimeric antibody is administered tohuman, antibodies against the monoclonal antibody derived from non-humananimal are not almost induced and the half-life in blood is prolongedsix times [Proc. Natl. Acad. Sci. USA, 86: 4220 (1989)]. The humanCDR-grafted antibody is an antibody in which thecomplementarity-determining region (CDR) of a human antibody is replacedwith the CDR of an antibody derived from non-human animal [Nature, 321:522 (1986)]. It is reported that in the experiment using a monkey,immunogenicity is lowered by the human CDR-grafted antibody as comparedwith a mouse antibody, and the half-life in blood is prolonged four tofive times [J. Immunol., 147: 1352 (1991)].

Accordingly, when the chimeric antibody and the humanized antibody whichspecifically react with human VEGF receptor Flt-1, side effects aredecreased and the half-life in blood is prolonged because no antibodyagainst a monoclonal antibody derived from non-human animal is produced.Therefore, it is expected that these antibodies can treat diseases inwhich their morbid states progress by abnormal angiogenesis, such asproliferation or metastasis of solid tumors, arthritis in rheumatoidarthritis, diabetic retinopathy, retinopathy of prematurity andpsoriasis, and the like.

In addition, with the recent progress of protein engineering and geneengineering, the production of smaller antibody molecules, such as asingle chain antibody [Science, 242: 423 (1988)] and a disulfidestabilized antibody [Molecular Immunology, 32: 249 (1995)], have beentried. Since the single chain antibody and the disulfide stabilizedantibody have a molecular weight lower than a monoclonal antibody or ahumanized antibody, they are excellent in tissue transition property andclearance from blood and are applied to imaging and the like, acomposite thereof with a toxin was prepared, and therefore, thetreatment effect can be expected [Cancer Research, 55: 318 (1995)].Therefore, it is expected that these antibodies can treat diseases inwhich their morbid states progress by abnormal angiogenesis, such asproliferation or metastasis of solid tumors, arthritis in rheumatoidarthritis, diabetic retinopathy, retinopathy of prematurity andpsoriasis, and the like.

SUMMARY OF THE INVENTION

Concern has been directed toward the development of a method which isuseful for the diagnosis or treatment of diseases in which their morbidstates progress by abnormal angiogenesis, such as proliferation ormetastasis of solid tumors, arthritis in rheumatoid arthritis, diabeticretinopathy, retinopathy of prematurity and psoriasis. Although nothinghas been reported on the anti-human VEGF receptor Flt-1 monoclonalantibody, it is considered that detection of the regions of angiogenesisand inhibition of angiogenesis by the use of an anti-human VEGF receptorFlt-1 monoclonal antibody will be useful for the diagnosis and treatmentof these diseases.

The above and other objects of the present invention may be accomplishedby a monoclonal antibody, that is, an antibody produced by a hybridoma,a humanized antibody, such as a human chimeric antibody and a humanCDR-grafted antibody, and an antibody fragment, such as Fab, Fab′,F(ab′)₂, single chain Fv, a disulfide stabilized antibody, and a peptidecomprising CDR, which each specifically reacts with human VEGF receptorFlt-1, preferably which each recognizes an epitope present in a regionof the 1st to 750th, more preferably 1st to 338th, and most preferably100th to 204th, positions from the N-terminal amino acid including asignal sequence of human VEGF receptor Flt-1 signal and receptorprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing construction steps of plasmid pVL1393/Flt 3N.

FIG. 2 is a graph showing construction steps of plasmid pVL1393/Flt 7N.

FIG. 3 is a graph showing patterns of SDS polyacrylamide electrophoresis(a 5 to 20% gradient gel was used) of purified Flt-1 7N and Flt-1 3N.Starting from the left side, electrophoresis patterns of molecularweight markers, Flt-1 3N and Flt-1 7N are shown respectively. Theelectrophoresis was carried out under reducing conditions.

FIG. 4 is a graph showing results of the analysis of the effect ofsoluble human VEGF receptors Flt-1 7N and Flt-1 3N to inhibit binding ofa ¹²⁵I-human VEGF to a plate-coated soluble human VEGF receptor Flt-17N.

FIG. 5 is a graph showing results of the examination of the bindingreactivity of anti-human VEGF receptor Flt-1 monoclonal antibody byenzyme immunoassay.

FIG. 6 is a graph showing results of the examination of the activity ofanti-human VEGF receptor Flt-1 monoclonal antibody to inhibit binding ofVEGF to human VEGF receptor Flt-1.

FIG. 7 is a graph showing results of the examination of the activity ofanti-human VEGF receptor Flt-1 monoclonal antibodies KM1732, KM1748 andKM1750 to inhibit binding of human VEGF to human VEGF receptor Flt-1.

FIG. 8 is a graph showing results of the examination of the activity ofanti-human VEGF receptor Flt-1 monoclonal antibodies KM1732, KM1748 andKM1750 to inhibit binding of human VEGF to human VEGF receptorFlt-1-expressing cells.

FIG. 9 is a graph showing results of the flow cytometry analysis of thereactivity of anti-human VEGF receptor Flt-1 monoclonal antibodiesKM1730, KM1731, KM1732, KM1748 and KM1750 with human VEGF receptorFlt-1-expressing cells NIH3T3-Flt-1 and control cells NIH3T3-Neo cells.

FIG. 10 is a graph showing results of the examination of the reactivityof anti-human VEGF receptor Flt-1 monoclonal antibody KM1737 with humanVEGF receptor Flt-1 by Western blotting. Lane 1 shows Western blottingpattern of NIH3T3-Flt-1 cells and lane 2 shows the pattern of NIH3T3-Neocells.

FIG. 11 is a graph showing results of the examination of thedetermination system of soluble human VEGF receptors Flt-1 3N and Flt-17N, carried out using anti-human VEGF receptor Flt-1 monoclonalantibodies KM1732 and biotinated KM1730.

FIG. 12 is a graph showing results of the flow cytometry analysis of thereactivity of anti-human VEGF receptor Flt-1 monoclonal antibody withhuman vascular endothelial cells HUVEC.

FIG. 13 is a graph showing results of the flow cytometry analysis of thereactivity of anti-human VEGF receptor. Flt-1 monoclonal antibody withhuman vascular endothelial cells HUVEC under a VEGF non-stimulation orstimulation condition.

FIG. 14 is a graph showing results of the analysis on the changes in theexpression quantity of human VEGF receptor Flt-1 in human vascularendothelial cells HUVEC under a VEGF non-stimulation or stimulationcondition. The expression quantity of Flt-1 is shown as a relativereaction value of anti-human VEGF receptor Flt-1 monoclonal antibodyKM1730 when the reactivity of a control antibody is defined as 1.

FIG. 15 is a graph showing construction steps of plasmid pBS1732H.

FIG. 16 is a graph showing construction steps of plasmid pBS1732L.

FIG. 17 is a graph showing construction steps of plasmid pKANTEX1732H.

FIG. 18 is a graph showing construction steps of plasmid pKANTEX1732.

FIG. 19 is a graph showing construction steps of plasmid pBS1750H.

FIG. 20 is a graph showing construction steps of plasmid pBS1750L.

FIG. 21 is a graph showing construction steps of plasmid pKANTEX1750H.

FIG. 22 is a graph showing construction steps of plasmid pKANTEX1750.

FIG. 23 is a graph showing SDS-PAGE (a 5 to 20% gradient gel was used)electrophoresis patterns of purified anti-human VEGF receptor Flt-1human chimeric antibodies KM2532 and KM2550 (lane 1: low molecularweight marker, lane 2: KM2532 under reducing conditions, lane 3: KM2550under reducing conditions, lane 4: high molecular weight marker, lane 5:KM2532 under non-reducing conditions, lane 6: KM2550 under non-reducingconditions).

FIG. 24 is a graph showing the activity of purified anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 to bind tosoluble human VEGF receptor Flt-1 7N. The upper drawing (A) shows theresults when concentration of soluble human VEGF receptor Flt-1 7N to beadsorbed on the plate is fixed (1 μg/ml) and concentration of each humanchimeric antibody to be added is varied. The binding activity withsoluble human VEGF receptor Flt-1 7N is plotted as ordinate and theconcentration of each human chimeric antibody as abscissa. The symbol Δindicates binding activity of KM2532 and ◯ indicates that of KM2550. Thelower drawing (B) shows the results when binding activity of each humanchimeric antibody at a fixed concentration (10 μg/ml) was measured byvarying concentration of soluble human VEGF receptor Flt-1 7N to beadsorbed on the plate. The binding activity with soluble human VEGFreceptor Flt-1 7N is plotted as ordinate, and the concentration ofsoluble human VEGF receptor Flt-1 7N adsorbed on the plate as abscissa.The symbol Δ indicates binding activity of KM2532 and ◯ indicates thatof KM2550.

FIG. 25 is a graph showing the activity of purified anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 to inhibitbinding between human VEGF and human VEGF receptor Flt-1. The symbols ◯,□, ● and ▪ shows the binding inhibition activities of KM1732, KM1750,KM2532 and KM2550, respectively.

FIG. 26 is a graph showing construction steps of plasmid phKM1732HV0.

FIG. 27 is a graph showing construction steps of plasmid phKM1750HV0.

FIG. 28 is a graph showing construction steps of plasmid phKM1732LV0.

FIG. 29 is a graph showing construction steps of plasmid phKM1750LV0(IV).

FIG. 30 is a graph showing construction steps of plasmid pKANTEX1732HV0.

FIG. 31 is a graph showing construction steps of plasmidpKANTEX1732HV0LV0.

FIG. 32 is a graph showing construction steps of plasmidpKANTEX1750HV0LV0 (IV).

FIG. 33 is a graph showing results of the enzyme immunoassay evaluationof the binding reactivity of anti-human VEGF receptor Flt-1 monoclonalantibodies KM1732 and KM1750 and anti-human VEGF receptor Flt-1 humanchimeric antibodies KM2532 and KM2550.

FIG. 34 is a graph showing results of the enzyme immunoassay evaluationof the binding reactivity of anti-human VEGF receptor Flt-1 humanchimeric antibodies KM2532 and KM2550.

FIG. 35 is a schematic illustration of soluble human VEGF receptorderivatives.

FIG. 36 is a graph showing construction steps of plasmid phKM1750LV0(I).

FIG. 37 is a graph showing construction steps of plasmidpKANTEX1750HV0LV0 (I).

FIG. 38 is a graph showing construction steps of plasmid phKM1750HV3.

FIG. 39 is a graph showing construction steps of plasmid phKM1750LV4.

FIG. 40 is a graph showing construction steps of plasmidpKANTEX1750HV3LV0 (I).

FIG. 41 is a graph showing construction steps of plasmidpKANTEX1750HV3LV0 (IV).

FIG. 42 is a graph showing construction steps of plasmidpKANTEX1750HV0LV4.

FIG. 43 is a graph showing construction steps of plasmidpKANTEX1750HV3LV4.

FIG. 44 is a graph showing SDS-PAGE (a 5 to 20% gradient gel was used)electrophoresis patterns of purified anti-human VEGF receptor Flt-1human chimeric antibody KM2550 and human CDR-grafted antibodies KM8550,KM8551, KM8552, KM8553, KM8554 and KM8555. Lanes 1 to 9 showelectrophoresis patterns under non-reducing conditions and lanes 10 to19 shows electrophoresis patterns under reducing conditions.

FIG. 45 is a graph showing the binding activity of purified anti-humanVEGF receptor Flt-1 human chimeric antibody KM2550 and human CDR-graftedantibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555 uponsoluble human VEGF receptor Flt-1 7N.

FIG. 46 is a graph showing the binding activity of purified anti-humanVEGF receptor Flt-1 human chimeric antibody KM2550 and human CDR-graftedantibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555 uponsoluble human VEGF receptor derivatives Flt-1 7N, Flt-1 3N, Flt-1 2N,Flt-1 7N.K2 and KDR-7N.

FIG. 47 is a graph showing the activity of purified anti-human VEGFreceptor Flt-1 human chimeric antibody KM2550 and human CDR-graftedantibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555 to inhibitthe binding between human VEGF and human VEGF receptor Flt-1.

FIG. 48 is a graph showing amino acid sequences of the H chain ofanti-human VEGF receptor Flt-1 human chimeric antibody and CDR-graftedantibody. In the drawing, KM1750mouse shows H chain V region amino acidsequence of KM1750; KM1750HV0 shows an amino acid sequence constitutedby inserting CDR of H chain V region of KM1750 into a human framework;and KM1750HV3 shows an amino acid sequence in which some of the aminoacid sequence of the framework of KM1750HV0 are substituted with theamino acids of KM1750mouse.

FIG. 49 is a graph showing amino acid sequences of L chain of anti-humanVEGF receptor Flt-1 human chimeric antibody and CDR-grafted antibody. Inthe drawing, KM1750 mouse shows an amino acid sequence of L chain Vregion of KM1750; KM1750LV0 (I) and KM1750LV0 (IV) each shows an aminoacid sequence constituted by inserting CDR of L chain V region of KM1750into a human framework; and KM1750LV4 shows an amino acid sequence inwhich some of the amino acid sequence of the framework of KM1750LV0 (I)are substituted with the amino acids of KM1750 mouse.

FIG. 50 is a graph showing results of the comparison of activities ofanti-VEGF receptor Flt-1 monoclonal antibodies KM1750 and KM1732 toinhibit migration of vascular endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a monoclonal antibody whichspecifically reacts with human VEGF receptor Flt-1; a monoclonalantibody which recognizes an epitope present in a region of amino acids1 to 750, preferably 1 to 338, and more preferably 100 to 204, of theN-terminal sequence of human VEGF receptor Flt-1 including a signalsequence; a monoclonal antibody which specifically reacts with humanVEGF receptor Flt-1 by immunocyte staining; a monoclonal antibody whichinhibits binding of human VEGF to human VEGF receptor Flt-1 and inhibitsbiological activities of human VEGF; and a monoclonal antibody whichinhibits migration of vascular endothelial cells.

Furthermore, the present invention relates to monoclonal antibody KM1730belonging to mouse IgG1 subclass produced by hybridoma KM1730 (FERMBP-5697); monoclonal antibody KM1731 belonging to mouse IgG2a subclassproduced by hybridoma KM1731 (FERM BP-5718); monoclonal antibody KM1732belonging to mouse IgG1 subclass produced by hybridoma KM1732 (FERMBP-5698); monoclonal antibody KM1748 belonging to mouse IgG2b subclassproduced by hybridoma KM1748 (FERM BP-5699); and monoclonal antibodyKM1750 belonging to mouse IgG2b subclass produced by hybridoma KM1750(FERM BP-5700).

Moreover, the present invention relates to hybridoma KM1730 (FERMBP-5697) which produces monoclonal antibody KM1730; hybridoma KM1731(FERM BP-5718) which produces monoclonal antibody KM1731; hybridomaKM1732 (FERM BP-5698) which produces monoclonal antibody KM1732;hybridoma KM1748 (FERM BP-5699) which produces monoclonal antibody ofKM1748; and hybridoma KM1750 (FERM BP-5700) which produces monoclonalantibody KM1750.

Also, the present invention relates to a treating or diagnostic agent ofdiseases in which their morbid states progress by abnormal angiogenesis,such as proliferation or metastasis of solid tumors, arthritis inrheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity,psoriasis, and the like.

The inventors of the present invention have found that anti-human VEGFreceptor Flt-1 monoclonal antibody capable of recognizing an epitopepresent in a region of the 1st to 750th positions from the N-terminalamino acid of human VEGF receptor Flt-1 can specifically react with thehuman VEGF receptor Flt-1 by immunocyte staining, and that biologicalactivities of human VEGF can be inhibited by the inhibition of bindingof VEGF to VEGF receptor Flt-1. Diagnosis and treatment of theabove-described diseases in which their morbid states progress byabnormal angiogenesis, such as proliferation or metastasis of solidtumors, arthritis in rheumatoid arthritis, diabetic retinopathy,prematurity retinopathy and psoriasis, can be carried out by using thesemonoclonal antibodies.

Consequently, the present invention provides antibodies whichspecifically react with human VEGF receptor Flt-1. With regard to themonoclonal antibody of the present invention, a monoclonal antibody isprovided that recognizes an epitope which is present in a region of the1st to 750th, preferably 1st to 338th, and more preferably 100th to204th, positions from the N-terminal amino acid including a signalsequence of human VEGF receptor Flt-1, and also specifically reacts withhuman VEGF receptor Flt-1 by immunocyte staining. Also, the presentinvention provides a monoclonal antibody which inhibits binding of humanVEGF to human VEGF receptor Flt-1 and also inhibits biologicalactivities of the human VEGF. Examples of the monoclonal antibody whichrecognizes the epitope and also specifically reacts with human VEGFreceptor Flt-1 by immunocyte staining include monoclonal antibody KM1730produced by the hybridoma KM1730 (FERM BP-5697), monoclonal antibodyKM1731 produced by the hybridoma KM1731 (FERM BP-5718), monoclonalantibody KM1732 produced by the hybridoma KM1732 (FERM BP-5698),monoclonal antibody KM1748 produced by the hybridoma KM1748 (FERMBP-5699), and monoclonal antibody KM1750 produced by the hybridomaKM1750 (FERM BP-5700). Examples of the monoclonal antibody whichinhibits binding of human VEGF to human VEGF receptor Flt-1 and alsoinhibits biological activities of human VEGF include monoclonal antibodyKM1732 produced by the hybridoma KM1732 (FERM BP-5698), monoclonalantibody KM1748 produced by the hybridoma KM1748 (FERM BP-5699), andmonoclonal antibody KM1750 produced by the hybridoma KM1750 (FERMBP-5700).

The monoclonal antibody of the present invention may be any antibody, solong as it specifically reacts with human VEGF receptor Flt-1. Examplesof the monoclonal antibody include an antibody produced by a hybridomaand a recombinant antibody produced by a transformant transformed withan expression vector containing the antibody gene. For example, thosewhich are established by the following production method can be cited aspreferred examples. That is, anti-human VEGF receptor Flt-1 monoclonalantibody can be obtained by preparing human VEGF receptor Flt-1 proteinas an antigen, immunizing an animal capable of providing a hybridomawith the antigen, such as mouse, rat, hamster, rabbit or the like,thereby inducing plasma cells having the antigen specificity, preparinga hybridoma capable of producing the monoclonal antibody through fusionof the cells with a myeloma cell line and subsequently culturing thehybridoma.

The monoclonal antibody which specifically reacts with human VEGFreceptor Flt-1 of the present invention may be a recombinant antibody.Examples of the recombinant antibody includes a humanized antibody andan antibody fragment.

The recombinant antibody of the present invention can be obtained bymodifying the above-described monoclonal antibody of the presentinvention using gene recombination technique. The recombinant antibodyincludes antibodies produced by gene recombination, such as a humanizedantibody and an antibody fragment (e.g., single chain antibody,disulfide stabilized antibody). Among these, antibodies which have thecharacteristics of monoclonal antibodies, show low antigenicity and haveprolonged half-life in blood are preferred as therapeutic agents.

The humanized antibody of the present invention includes a humanchimeric antibody and a human CDR (complementarity-determining region;hereinafter referred to as “CDR”)-grafted antibody.

The antibody fragment of the present invention includes a fragment ofantigen binding (hereinafter referred to as “Fab”), Fab′, F(ab′)₂, asingle chain antibody (single chain Fv; hereinafter referred to as“scFv”), and a disulfide stabilized antibody (disulfide stabilized Fv;hereinafter referred to as “dsFv”), which specifically react with humanVEGF receptor Flt-1. Hereinafter, the antibody which specifically reactswith human VEGF receptor Flt-1 is referred to as an “anti-human VEGFreceptor Flt-1 antibody”. Furthermore, the antibody fragments of thepresent invention are included within the scope of the antibodies of thepresent invention.

The antibody which reacts with human VEGF receptor Flt-1 of the presentinvention may be a humanized antibody which is selected from a humanchimeric antibody and a human CDR-grafted antibody.

The humanized antibody of the present invention may comprise CDR of anantibody heavy chain (H chain) variable region (V region) comprisingamino acid sequences of from 31 to 35, from 50 to 66, and from 99 to 108positions (SEQ ID NOS:9, 10 and 11, respectively) described in SEQ IDNO:86 (corresponding to nucleic acid sequence of SEQ ID NO:5) or aminoacid sequences of from 31 to 35, from 50 to 66, and from 99 to 106positions (SEQ ID NOS:15, 16 and 17, respectively) described in SEQ IDNO:88 (corresponding to nucleic acid sequence of SEQ ID NO:7). Also, thehumanized antibody may comprise CDR of an antibody light chain (L chain)variable region (V region) comprising amino acid sequences of from 24 to34, from 49 to 55, and from 88 to 96 positions (SEQ ID NO:12, 13 and 14,respectively) described in SEQ ID NO:87 (corresponding to nucleic acidsequence of SEQ ID NO:6) or amino acid sequences of from 24 to 33, from49 to 55, and from 88 to 94 positions (SEQ ID NOS:15, 16 and 17,respectively) described in SEQ ID NO:89 (corresponding to nucleic acidsequence of SEQ ID NO:8).

The human chimeric antibody of the present invention may comprise anantibody H chain V region and an antibody L chain V region of amonoclonal antibody which specifically reacts with human VEGF receptorFlt-1, and H chain constant region (C region) and L chain C region of ahuman antibody.

Specifically, the human chimeric antibody means an antibody whichcomprises V region H chain (hereinafter referred to as “VH”) and Vregion L chain (hereinafter referred to as “VL”) of an antibody of anon-human animal, C region H chain (hereinafter referred to as “CH”) ofa human antibody, and C region L chain (hereinafter referred to as “CL”)of a human antibody.

The human chimeric antibody of the present invention can be produced bypreparing cDNAs encoding VH and VL from a hybridoma capable of producinga monoclonal antibody which specifically reacts with human VEGF receptorFlt-1, inserting them into an expression vector for animal cells havinggenes encoding human antibody CH and human antibody CL to construct ahuman chimeric antibody expression vector, and then introducing thevector into cells of an animal to express the antibody of interest.

The structure of the human chimeric antibody of the present inventionmay belong to any immunoglobulin (Ig) class, but preferably contains theC region of IgG type immunoglobulin, particularly of IgG subclasses,such as IgG1, IgG2, IgG3, and IgG4.

The H chain V region and the L chain V region of the human chimericantibody of the present invention may comprise an amino acid sequence ofH chain V region and L chain V region, respectively, of a monoclonalantibody selected from the group consisting of monoclonal antibodyKM1732 (FERM BP-5698) and monoclonal antibody KM1750 (FERM BP-5700).Also, in the human chimeric antibody of the present invention, the aminoacid sequence of H chain V region may be an amino acid sequence of SEQID NO:86 or 88, and the amino acid sequence of L chain V region may bean amino acid sequence of SEQ ID NO:87 or 89.

An example of the human chimeric antibody of the present inventionwherein the amino acid sequence of H chain V region is an amino acidsequence of SEQ ID NO:86, and the amino acid sequence of L chain Vregion is an amino acid sequence of SEQ ID NO:87 includes KM2532. Also,an example of the human chimeric antibody of the present inventionwherein the amino acid sequence of E chain V region is an amino acidsequence of SEQ ID NO:88, and the amino acid sequence of L chain Vregion is an amino acid sequence of SEQ ID NO:89 includes KM2550.

In addition, the present invention includes a DNA which encodes any oneof the above human chimeric antibodies which specifically react withhuman VEGF receptor Flt-1.

According to the present invention, a recombinant vector comprising theabove DNA and tandem cassette vector pKANTEX93 can be provided; atransformant can be obtained by introducing the recombinant vector intoa host cell; and the human chimeric antibody can be provided culturingthe transformant in a medium to produce and accumulate a human chimericantibody in a culture, and recovering the antibody from the resultingculture.

The anti-human VEGF receptor Flt-1 antibody may be a humanized antibodywhich is a human CDR-grafted antibody.

The human CDR-grafted antibody of the present invention means anantibody in which the CDRs of VH and VL of a human antibody are replacedwith respective CDRs of an antibody of a non-human animal.

The human CDR-grafted antibody of the present invention can be producedby constructing V region-encoding cDNAs in which CDRs of VH and VL of ahuman antibody are replaced with CDRs of VH and VL of an antibody of anon-human animal, which specifically reacts with human VEGF receptorFlt-1, inserting them into an expression vector for animal cells havinggenes encoding human antibody CH and human antibody CL to construct ahuman CDR-grafted antibody expression vector, and then introducing thevector into cells of an animal to express the antibody of interest.

The structure of the C region of the human CDR-grafted antibody of thepresent invention may belong to any immunoglobulin (Ig) class, butpreferably contains the C region of IgG type immunoglobulin,particularly of IgG subclasses such as IgG1, IgG2, IgG3 and IgG4.

The human CDR-grafted antibody of the present invention may comprise Vregion complementarity-determining regions (CDRs) of H chain and L chainof a monoclonal antibody capable of specifically reacting with humanVEGF receptor Flt-1, and C region and V region framework regions of Hchain and L chain of a human antibody.

In the human CDR-grafted antibody of the present invention, the CDR of Hchain V region of the human CDR-grafted antibody may comprise amino acidsequences of SEQ ID NOS:9, 10 and 11 or amino acid sequences of SEQ IDNOS:15, 16 and 17; and the CDR of L chain V region of the humanCDR-grafted antibody may comprise amino acid sequences of SEQ ID NOS:12,13 and 14 or amino acid sequences of SEQ ID NOS:18, 19 and 20.

Also, examples of the human CDR-grafted antibody of the presentinvention include a human CDR-grafted antibody wherein the CDR of Hchain V region of the human CDR-grafted antibody comprises amino acidsequences of SEQ ID NOS:9, 10 and 11, and the CDR of L chain V region ofthe human CDR-grafted antibody comprises amino acid sequences of SEQ IDNOS:12, 13 and 14; and a human CDR-grafted antibody wherein the CDR of Hchain V region of the human CDR-grafted antibody comprises amino acidsequences of SEQ ID NOS:15, 16 and 17, and the CDR of L chain V regionof the human CDR-grafted antibody comprises amino acid sequences of SEQID NOS:18, 19 and 20.

Further examples of the human CDR-grafted antibody of the presentinvention include a human CDR-grafted antibody wherein the H chain Vregion of the human CDR-grafted antibody comprises an amino acidsequence of SEQ ID NO:90, and the L chain V region of the humanCDR-grafted antibody comprises an amino acid sequence of SEQ ID NO:92;and a human CDR-grafted antibody wherein the H chain V region of thehuman CDR-grafted antibody comprises an amino acid sequence of SEQ IDNO:91 or 95, and the L chain V region of the human CDR-grafted antibodycomprises an amino acid sequence of SEQ ID NO:93, 94 or 96.

Specific examples of the human CDR-grafted antibody of the presentinvention include human CDR-grafted antibody KM8550, human CDR-graftedantibody KM8551, human CDR-grafted antibody KM8552, human CDR-graftedantibody KM8553, human CDR-grafted antibody KM8554, and humanCDR-grafted antibody KM8555.

In addition, the present invention includes a DNA which encodes any oneof the human CDR-grafted antibodies.

According to the present invention, a recombinant vector comprising theabove DNA and tandem cassette vector pKANTEX93 can be provided; atransformant can be obtained by introducing the recombinant vector intoa host cell; and the human CDR-grafted antibody can be prepared byculturing the transformant in a medium to produce and accumulate aCDR-grafted antibody in a culture, and recovering the antibody from theresulting culture.

The anti-human VEGF receptor Flt-1 antibody of the present invention maybe an antibody fragment selected from the group consisting of Fab, Fab′,F(ab′)₂, scFv, and dsFv.

The Fab is a fragment having a molecular weight of about 50,000 andantigen-binding activity which comprises about half of the N-terminalside of H chain and a full portion of L chain obtained by digesting,with papain, the peptide moiety of the upper side of two disulfide bondsthat cross-link two H chains at the hinge region of IgG.

The Fab of the present invention can be obtained by treating ananti-human VEGF receptor Flt-1 antibody with papain. Alternatively, theFab can be produced by inserting a DNA fragment which encodes the Fabfragment of the antibody into an expression vector for animal cells, andintroducing the vector into cells of an animal to express the antibodyof interest.

The Fab′ is a fragment of about 50,000 in molecular weight havingantigen-binding ability which is obtained by hydrolyzing the disulfidebond between hinges of the above-described F(ab′)₂.

The Fab′ of the present invention can be obtained by treating ananti-human VEGF receptor Flt-1 antibody with a reducing agent such asdithiothreitol. Alternatively, the Fab′ can be produced by inserting aDNA fragment which encodes the Fab′ fragment of the antibody into anexpression vector for animal cells, and introducing the vector intocells of an animal to express the antibody of interest.

The F(ab′)₂ of the present invention is a fragment having a molecularweight of about 100,000 and antigen-binding ability, comprising two Fabregions bonded at the hinge part, which is obtained by hydrolyzing, withtrypsin, the lower side of two disulfide bonds at the hinge region ofIgG.

The F(ab′)₂ of the present invention can be obtained by treating ananti-human VEGF receptor Flt-1 antibody with trypsin. Alternatively,F(ab′)₂ can be produced by inserting a DNA fragment which encodes theF(ab′)₂ fragment of the antibody into an expression vector for animalcells, and introducing the vector into cells of an animal to express theantibody of interest.

The anti-human VEGF receptor Flt-1 antibody of the present invention maybe scFv comprising H chain V region and L chain V region of an antibody.

The scFv of the present invention means a VH-P-VL or VL-P-VH polypeptideobtained by linking a VH chain with a VL chain using an appropriatepeptide linker (hereinafter referred to as “P”). The VH and VL of thescFv of the present invention may be any of the monoclonal antibody andhuman CDR-grafted antibody of the present invention.

The scFv of the present invention can be prepared by preparing cDNAsencoding VH and VL from a hybridoma capable of producing an anti-humanVEGF receptor Flt-1 antibody to construct an scFv expression vector,inserting the cDNAs into the scFv expression vector, and introducing theexpression vector into cells of Escherichia coli, a yeast or an animalto express the antibody of interest.

In the scFv of the present invention, the H chain V region and the Lchain V region of the scFv may comprise an amino acid sequence which isthe same as an amino acid sequence of H chain V region and L chain Vregion, respectively, of a monoclonal antibody which specifically reactswith human VEGF receptor Flt-1; or the H chain V region and the L chainV region of the scFv may comprise CDR comprising an amino acid sequencewhich is the same as an amino acid sequence of CDR of H chain V regionand L chain V region, respectively, of a monoclonal antibody whichspecifically reacts with human VEGF receptor Flt-1.

Also, in the scFv of the present invention, the H chain V region and theL chain V region of the scFv may comprise an amino acid sequences of SEQID NOS:86 and 87, respectively; or the H chain V region and the L chainV region of the scFv may comprise an amino acid sequence of SEQ IDNOS:88 and 89, respectively.

Examples of the scFv of the present invention include scFv wherein theCDR of the H chain V region of the scFv comprises amino acid sequencesof SEQ ID NOS:9, 10 and 11, and the CDR of the L chain V region of thescFv comprises amino acid sequences of SEQ ID NOS:12, 13 and 14; andscFv wherein the CDR of the H chain V region of the scFv comprises aminoacid sequences of SEQ ID NOS:15, 16 and 17, and the CDR of the L chain Vregion of the scFv comprises amino acid sequences of SEQ ID NOS:18, 19and 20.

Further examples of the scFv of the present invention include scFvwherein the H chain V region of the scFv comprises an amino acidsequence of SEQ ID NO:90, and the L chain V region of the scFv comprisesan amino acid sequence of SEQ ID NO:92; and scFv wherein the H chain Vregion of the scFv comprises an amino acid sequence of SEQ ID NO:91 or95, and the L chain V region of the scFv comprises an amino acidsequence of SEQ ID NO:93, 94 or 96.

The anti-human VEGF receptor Flt-1 antibody may be dsFv comprising Hchain V region and L chain V region of an antibody.

The dsFv means an antibody obtained by bonding, via a disulfide bond,polypeptides in which one amino acid residue in each of VH and VL isreplaced with a cysteine residue. The amino acid residue to be replacedwith a cysteine residue can be selected based on the three-dimensionalstructure estimation of antibodies in accordance with the methodreported by Reiter et al. [Protein Engineering, 7: 697 (1994)]. The VHor VL contained in the dsFv of the present invention may be any of themonoclonal antibody and human CDR-grafted antibody of the presentinvention.

The dsFv of the present invention can be prepared by preparing cDNAsencoding VH and VL from a hybridoma capable of producing an anti-humanVEGF receptor Flt-1 antibody, inserting the cDNAs into an appropriateexpression vector, introducing the expression vector into cells ofEscherichia coli, a yeast, an animal or the like to express the antibodyof interest.

In the dsFv of the present invention, the H chain V region and the Lchain V region of the dsFv may comprise an amino acid sequence which isthe same as an amino acid sequence of H chain V region and L chain Vregion, respectively, of a monoclonal antibody which specifically reactswith human VEGF receptor Flt-1; or the H chain V region and the L chainV region of the sdFv may comprise CDR comprising an amino acid sequencewhich is the same as an amino acid sequence of CDR of H chain V regionand L chain V region, respectively, of a monoclonal antibody whichspecifically reacts with human VEGF receptor Flt-1.

Also, in the dsFv of the present invention, the H chain V region and theL chain V region of the dsFv may comprise an amino acid sequence of SEQID NOS:86 and 87, respectively; or the H chain V region and the L chainV region of the dsFv may comprise an amino acid sequence which is thesame as an amino acid sequence of SEQ ID NOS:88 and 89, respectively.

Examples of the dsFv of the present invention include dsFv wherein theCDR of the H chain V region of the dsFv comprises amino acid sequencesof SEQ ID NOS:9, 10 and 11, and the CDR of the L chain V region of thedsFv comprises amino acid sequences of SEQ ID NOS:12, 13 and 14; anddsFv wherein the CDR of the H chain V region of the dsFv comprises aminoacid sequences of SEQ ID NOS:15, 16 and 17, and the CDR of the L chain Vregion of the dsFv comprises amino acid sequences of SEQ ID NOS:18, 19and 20.

Further examples of the dsFv of the present invention include dsFvwherein the H chain V region of the dsFv comprises an amino acidsequence of SEQ ID NO:90, and the L chain V region of the dsFv comprisesan amino acid sequence of SEQ ID NO:92; and dsFv wherein the H chain Vregion of the dsFv comprises an amino acid sequence of SEQ ID NO:91 or95, and the L chain V region of the dsFv comprises an amino acidsequence of SEQ ID NO:93, 94 or 96.

Still furthermore, the present invention relates to a peptide comprisingamino acids selected from CDRs of H chain and L chain of an antibody,which specifically reacts with human VEGF receptor Flt-1, such as apeptide comprising an amino acid sequence selected from amino acidsequences of SEQ ID NOS:9, 10, 11, 12, 13 and 14, and a peptidecomprising an amino acid sequence selected from amino acid sequences ofSEQ ID NOS:15, 16, 17, 18, 19 and 20.

Still moreover, the present invention relates to a fusion antibody or afusion peptide which is an antibody or a peptide chemically orgene-engineeringly linked with a radioisotope, a protein or a lowmolecular agent.

The fusion antibody and the fusion peptide of the present invention canbe produced by chemically linking a radioactive isotope, a protein or alow molecular agent with the antibody or peptide which specificallyreacts with human VEGF receptor Flt-1 of the present invention. In thecase of a fusion antibody or a fusion peptide with a protein, it can beproduced by connecting an antibody- or a peptide-encoding cDNA to aprotein-encoding cDNA, inserting the thus ligated cDNA into anappropriate expression vector, and expressing the expression vector incells of Escherichia coli, a yeast, an animal or the like.

In addition, the present invention relates to the following methods:

a method for immunologically detecting human VEGF receptor Flt-1,comprising reacting human VEGF receptor Flt-1 with the antibody orpeptide of the present invention;

a method for immunologically detecting cells in which human VEGFreceptor Flt-1 is expressed on the surface thereof, comprising reactinghuman VEGF receptor Flt-1 with the antibody or peptide of the presentinvention;

a method for inhibiting binding of human VEGF to human VEGF receptorFlt-1, comprising reacting human VEGF receptor Flt-1 with the antibodyor peptide of the present invention;

a method for inhibiting biological activities of human VEGF, comprisingreacting human VEGF receptor Flt-1 with the antibody or peptide of thepresent invention;

a method for detecting a disease in which the morbid states progress byabnormal angiogenesis, comprising reacting a sample with the antibody orpeptide of the present invention; and

a method for preventing or treating a disease, comprising the step ofadministering to human or animal in need of such prevention or treatmentan effective amount of the antibody or peptide of the present invention.

In the above method for immunologically detecting human VEGF receptorFlt-1, the human VEGF receptor Flt-1 may be soluble.

In the above method for inhibiting biological activities of human VEGF,for example, the activity of human VEGF receptor Flt-1 is inhibited.

In the above method for detecting a disease, for example, the method maycomprise (a) separating human cell or a crushing solution thereof,tissue or a crushing solution thereof, serum, pleural fluid, ascitesfluid, or ocular fluid to prepare a sample, (b) reacting the separatedsample prepared in the step (a) with the monoclonal antibody or peptideof the present invention, (c) further reacting the reacted sampleprepared in the step (b) with a labeled anti-mouse IgG antibody orbinding fragment, and (d) measuring or observing the labeled sampleprepared in the step (c).

In the above method for preventing or treating a disease, examples ofthe disease include diseases in which the morbid states progress byabnormal angiogenesis.

Examples of the diseases in which their morbid states progress byabnormal angiogenesis include proliferation or metastasis of solidtumor, arthritis in chronic rheumatoid arthritis, diabetic retinopathy,retinopathy of prematurity, and psoriasis. Examples of the solid tumorinclude breast cancer, prostatic cancer, large bowel cancer, gastriccancer and lung cancer.

Besides, the present invention relates to a composition comprising theantibody or peptide of the present invention and a diagnostic orpharmaceutical acceptable carrier.

The present invention is discussed below in more detail.

The production method of the anti-human VEGF receptor Flt-1 antibody ofthe present invention is described below.

1. Production Method of Anti-human VEGF Receptor Flt-1 monoclonalAntibody

(1) Preparation of Antigen

Examples of the substance useful as the antigen for the preparation ofthe anti-human VEGF receptor Flt-1 monoclonal antibody include cells inwhich human VEGF receptor Flt-1 is expressed on the cell surface or acell membrane fraction thereof, soluble human VEGF receptor Flt-1protein having an extracellular region of different length and a fusionprotein of the protein with Fc region of the antibody. As the cellscapable of expressing human VEGF receptor Flt-1 on the cell surface,NIH3T3-Flt-1 cells [Oncogene, 10: 135 (1995)] can be exemplified. In amethod for expressing the antigen as soluble human VEGF receptor Flt-1protein having an extracellular region of different length or a fusionprotein of the protein with Fc region of the antibody, the whole lengthor a partial fragment of cDNA which encodes human VEGF receptor Flt-1[Oncogene, 5: 519 (1990); Abstract of Papers, the 18th Annual meeting ofJapan Molecular Biology Society, 2P-227 (December, 1995)] is insertedinto a downstream site of the promoter of an appropriate vector, thethus constructed recombinant vector is inserted into host cells and thethus obtained human VEGF receptor Flt-1 expression cells are cultured inan appropriate medium, thereby producing the whole length of a partialfragment of human VEGF receptor Flt-1 in the cells or culturesupernatant as such or as a fusion protein.

As the host cells, any one of bacteria, yeast, animal cells, insectcells and the like can be used so long as they can express the gene ofinterest. Examples of the bacteria include the genus Escherichia, thegenus Bacillus and the like, such as Escherichia coli, Bacillus subtilisand the like. Examples of the yeast include Saccharomyces cerevisiae,Schizosaccharomyces pompe and the like. Examples of the animal cellsinclude namalwa cells which are human cells, COS cells which are monkeycells and CHO cells which are Chinese hamster cells. Examples of theinsect cells include Sf9 and Sf21 (manufactured by Pharmingen), HighFive (manufactured by Invitrogen) and the like.

When a bacterium such as Escherichia coli is used as the host, theexpression vector may be preferably constructed with a promoter, aribosome binding sequence, the DNA of the present invention, atranscription termination sequence and, as occasion demands, a promotercontrolling sequence. Examples include commercially available pGEX(manufactured by Pharmacia), pET System (manufactured by Novagen) andthe like.

With regard to the method for introducing the recombinant vector into abacterium, any one of the known methods for introducing DNA intobacteria, such as a method in which calcium ion is used [Proc. Natl.Acad. Sci. USA, 69: 2110-2114 (1972)], a protoplast method (JapanesePublished Unexamined Patent Application No. 2483942/91), and the likecan be used.

When yeast is used as the host, YEp13 (ATCC 37115), YEp24 (ATCC 37051),YCp50 (ATCC 37419), or the like is used as the expression vector.

With regard to the method for introducing the recombinant vector intoyeast, any one of the known methods for introducing DNA into yeast, suchas an electroporation method [Methods. Enzymol., 194: 182-187 (1990)], aspheroplast method [Proc. Natl. Acad. Sci. USA, 84: 1929-1933 (1978)], alithium acetate method [J. Bacteriol., 153: 163-168 (1983)], and thelike can be used.

When animal cells are used as the host, pAGE107 [Japanese PublishedUnexamined Patent Application No. 22979/88; Cytotechnology, 3: 133(1990)], pAGE103 [J. Biochem., 101: 1307 (1987)], and the like can beexemplified as the useful expression vector.

Any promoter capable of carrying out the expression in animal cells canbe used. Examples include the promoter of IE (immediate early) gene ofcytomegalovirus (CMV), the SV40 promoter, the metallothionein promoterand the like. Furthermore, the enhancer of the IE gene of human CMV maybe used together with the promoter.

With regard to the method for the introduction of the recombinant vectorinto animal cells, any one of the known methods for introducing DNA intoanimal cells, such as an electroporation method [Cytotechnology, 3: 133(1990)], a calcium phosphate method (Japanese Published UnexaminedPatent Application No. 227075/90), a lipofection method [Proc. Natl.Acad. Sci. USA, 84: 7413 (1987)] and the like can be used.

When insect cells are used as the host, the protein can be expressed bythe known method described in, for example, Current Protocols inMolecular Biology, Supplement 1-34 and Baculovirus expression vectors, Alaboratory manual. That is, the recombinant gene introducing vector andbaculovirus described in the following are simultaneously introducedinto insect cells to obtain a recombinant virus in the insect cellculture supernatant and then the insect cells are infected with the thusobtained recombinant virus to obtain protein-expressing insect cells.

Examples of the gene introducing vector include pVL1392, pVL1393,pBlueBacIII (all manufactured by Invitrogen), and the like.

Examples of the baculovirus include Autograph californica nuclearpolyhedrosis virus with which insects of the family Barathra areinfected.

With regard to the method for the simultaneous introduction of theabove-described recombinant gene introducing vector and theabove-described baculovirus into insect cells for the preparation of therecombinant virus, calcium phosphate method (Japanese PublishedUnexamined Patent Application No. 227075/90), lipofection method [Proc.Natl. Acad. Sci. USA, 84: 7413 (1987)] and the like can be exemplified.

Alternatively, the protein of interest can be produced by preparing arecombinant baculovirus making use, for example, of BaculoGold StarterKit manufactured by Pharmigen and then infecting the above-describedinsect cells, such as Sf9, Sf21, High Five, or the like, with therecombinant virus [Bio/Technology, 6: 47 (1988)].

With regard to the gene expression method, techniques, such as secretionproduction, fusion protein expression and the like have been developed,and each of these every methods can be used. For example, geneexpression can be produced in accordance with the method described inMolecular Cloning 2nd edition, Cold Spring Harbor Lab. Press, New York(1989), or by direct expression.

The whole length or a partial fragment of a human VEGF receptor Flt-1can be produced as such or as a fusion protein thereof by culturing atransformant obtained in the above-described manner in a culture mediumto form and accumulate the protein of the present invention in theresulting culture mixture, and then collecting the protein from theculture mixture.

Culturing of the transformant of the present invention in a culturemedium is carried out in accordance with a usual method which is used inthe culturing of respective host cells.

With regard to the medium for use in the culturing of the transformantobtained using a microorganism, such as Escherichia coli, yeast, or thelike, as the host, either a natural medium or a synthetic medium may beused, so long as it contains materials which can be assimilated by themicroorganism, such as carbon sources, nitrogen sources, inorganicsalts, and the like, and can perform culturing of the transformantefficiently [Molecular Cloning 2nd edition, Cold Spring Harbor Lab.Press, New York (1989)]. The culturing is carried out generally underaerobic conditions, such as a shaking culture, submerged agitationaeration culture, or the like, at 15 to 40° C. for 16 to 96 hours.During the culturing, the pH is controlled to 3.0 to 9.0. Adjustment ofthe pH is carried out using an inorganic or organic acid, an alkalisolution, urea, calcium carbonate, ammonia, and the like. As occasiondemands, antibiotics, such as ampicillin, tetracycline, and the like maybe added to the medium during the culturing.

With regard to the medium for use in the culturing of a transformantobtained using animal cells as the host, RPMI 1640 medium, Eagle's MEMmedium or any one of these media further supplemented with fetal calfserum may be used. The culturing is carried out generally at 35 to 37°C. for 3 to 7 days in the presence of 5% CO₂. As occasion demands,antibiotics, such as kanamycin, penicillin, and the like may be added tothe medium during the culturing.

With regard to the medium for use in the culturing of a transformantobtained using insect cells as the host, TNM-FH medium (manufactured byPharmingen), Sf900IISFM (manufactured by Life Technologies), ExCell400or ExCell405 (both manufactured by JRH Biosciences), or the like may beused. The culturing is carried out generally at 25 to 30° C. for 1 to 4days, and gentamicin and the like antibiotics may be added to the mediumduring the culturing as occasion demands.

Although media for the culturing of animal cells and insect cellscontain serum, it is desirable to use a serum-free medium in order toefficiently purify the whole length or a partial fragment of human VEGFreceptor Flt-1 as such or as a fusion protein.

When the whole length or a partial fragment of human VEGF receptor Flt-1is accumulated inside the host cells as such or as a fusion protein, thecells after completion of the culturing are collected by centrifugation,suspended in an aqueous buffer and then disrupted using ultrasonicoscillator, French press, or the like, and subsequently collecting theprotein from a supernatant fluid prepared by centrifuging the thusdisrupted cells.

Also, when an insoluble body is formed inside the cells, the insolublebody is solubilized using a protein denaturing agent and thenhigher-order structure of the protein is formed by diluting or dialyzingthe thus solubilized protein in or against a solution which does notcontain the protein denaturing agent or contains the agent but in such alow concentration that the protein is not denatured.

When the whole length or a partial fragment of human VEGF receptor Flt-1is secreted outside the cells as such or as a fusion protein, theexpressed protein can be collected from the culture supernatant. Theisolation and purification can be carried out by employing separationmeans, such as solvent extraction, fractional precipitation with organicsolvents, salting out, dialysis, centrifugation, ultracentrifugation,ion exchange chromatography, gel filtration chromatography, hydrophobicchromatography, affinity chromatography, reverse phase chromatography,crystallization, electrophoresis, and the like, alone or in combination.

(2) Immunization of Animals and Preparation of Antibody Producing Cells

Although any one of animals, such as mice, rats, hamsters, rabbits, andthe like, can be used in the immunization, so long as a hybridoma can beprepared, an example in which mice and rats are used is described inthis invention. A mouse or rat of 3 to 20 weeks of age is immunized withthe protein obtained in the above step 1-(1) as the antigen, andantibody producing cells are collected from the spleen, lymph node orperipheral blood of the animal. The immunization is carried out byadministering the antigen several times through subcutaneous,intravenous or intraperitoneal injection together with an appropriateadjuvant. As the adjuvant, a complete Freund's adjuvant or a combinationof aluminum hydroxide gel with pertussis vaccine can be exemplified. Ablood sample is collected from the fundus of the eye or caudal vein ofthe animal 3 to 7 days after each administration, the sample is tested,for example, by enzyme immunoassay [Enzyme-linked Immunosorbent Assay(ELISA), published by Igaku Shoin, (1976)] as to whether it is reactivewith the antigen used, namely soluble human VEGF receptor Flt-1 orNIH3T3 cells in which human VEGF receptor Flt-1 is expressed on the cellsurface, and then a mouse or rat showing sufficient antibody titer intheir sera is submitted for use as the supply source of antibodyproducing cells. On the 3rd to 7th day after final administration of theantigen, the spleen is excised from the immunized mouse or rat to carryout fusion of the spleen cells with myeloma cells in accordance with theknown method [Antibodies—A Laboratory Manual, Cold Spring HarborLaboratory, (1988); referred to as “Antibodies—A Laboratory Manual”hereinafter].

(3) Preparation of Myeloma Cells

As the myeloma cells, any myeloma cells capable of growing in vitro maybe used, which include established cells obtained from mouse, such as8-azaguanine-resistant mouse (BALB/c) myeloma cell line P3-X63Ag8-U1(P3-U1) [G. Kohler et al., Europ. J. Immunol, 6: 511 (1976)], SP2/O—Ag14(SP-2) [M. Shulman et al., Nature, 276: 269 (1978)], P3-X63-Ag8653 (653)[J. F. Kearney et al., J. Immunol., 123: 1548 (1979)], P3-X63-Ag8 (X63)[G. Kohler et al., Nature, 256: 495 (1975)], and the like. These celllines are cultured and subcultured in accordance with the known method(Antibodies—A Laboratory Manual) and 2×10⁷ or more of the cells aresecured until cell fusion.

(4) Cell Fusion

The antibody producing cells obtained in the above step (2) and themyeloma cells obtained in the above step (3) are washed, mixed with cellaggregating medium, polyethylene glycol-1000 (PEG-1000) or the like, tocarry out cell fusion and then suspended in a culture medium. For thewashing of the cells, MEM medium or PBS (1.83 g of disodium hydrogenphosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g of sodiumchloride, 1 liter of distilled water, pH 7.2) is used. In order toobtain the fused cells of interest selectively, HAT medium {normalmedium [a medium prepared by adding glutamine (1.5 mM),2-mercaptoethanol (5×10⁻⁵ M), gentamicin (10 μg/ml) and fetal calf serum(FCS) (10%, produced by CSL) to RPMI-1640 medium] further supplementedwith hypoxanthine (10⁻⁴ M), thymidine (1.5×10⁻⁵ M) and aminopterin(4×10⁻⁷ M)} is used as the medium for suspending the fused cells.

After the culturing, a portion of the culture supernatant is sampled andtested, for example, by an enzyme immunoassay method which will bedescribed in the following step (5) to select wells which canspecifically react with human VEGF receptor Flt-1 or a recombinantprotein such as a fusion protein with human VEGF receptor Flt-1described in the above step (1). Thereafter, cloning is carried outtwice by limiting dilution analysis [using HT medium (a medium preparedby eliminating aminopterin from the HAT medium) for the first analysisand the normal medium for the second analysis], and a hybridoma whichshows stable and high antibody titer is selected as the hybridomacapable of producing the anti-human VEGF receptor Flt-1 monoclonalantibody.

(5) Selection of Anti-human VEGF Receptor Flt-1 Monoclonal Antibody

Selection of a hybridoma capable of producing the anti-human VEGFreceptor Flt-1 monoclonal antibody is carried out by the enzymeimmunoassay method described below.

Enzyme Immunoassay

Human VEGF receptor Flt-1 or a recombinant protein such as a fusionprotein with the human VEGF receptor Flt-1 described in the above step1-(1) is coated on an appropriate plate and allowed to react with afirst antibody, namely a hybridoma culture supernatant or a purifiedantibody obtained in the following step 1-(6), and then with a secondantibody, namely an anti-mouse immunoglobulin antibody or anti-ratimmunoglobulin antibody labeled with biotin, an enzyme, achemiluminescent substance, a radioactive compound or the like, and thena reaction suitable for the label used is carried out in order to selecta sample which specifically reacts with human VEGF receptor Flt-1 as ahybridoma capable of producing anti-human VEGF receptor Flt-1 monoclonalantibody. Examples of the hybridoma include hybridomas KM1730, KM1731,KM1732, KM1748 and KM1750. The hybridomas KM1730, KM1732, KM1748 andKM1750, on Oct. 8, 1996, and the hybridoma KM1731, on Oct. 22, 1996,were deposited with National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology (Higashi 1-1-3,Tsukuba-shi, Ibaraki, Japan), and were assigned the designations as FERMBP-5697, FERM BP-5698, FERM BP-5699, FERM BP-5700 and FERM BP-5718,respectively.

(6) Preparation of Monoclonal Antibody

The anti-human VEGF receptor Flt-1 monoclonal antibody-producinghybridoma cells obtained in the above-described step 1-(3) areadministered by intraperitoneal injection into 8- to 10-week-old mice ornude mice treated with pristane [by intraperitoneal administration of0.5 ml of 2,6,10,14-tetramethylpentadecane (pristane) followed by 2weeks of feeding] at a dose of 2×10⁷ to 5×10⁶ cells/animal. Thehybridoma causes ascites tumor in 10 to 21 days. The ascitic fluid iscollected from the mice or nude mice, centrifuged, subjected to saltingout with 40 to 50% saturated ammonium sulfate or to caprylic acidprecipitation and then passed through a DEAE-Sepharose column, protein Acolumn or Cellulofine GSL 2000 (manufactured by Seikagaku Kogyo) tocollect an IgG or IgM fraction to give a purified monoclonal antibody.

The subclass of the purified monoclonal antibody can be determined usinga mouse monoclonal antibody typing kit or a rat monoclonal antibodytyping kit. The amount of protein can be determined by the Lowry methodor by calculation based on the optical density at 280 nm.

The term “subclass of antibody” as used herein means isotypes within theclass, such as IgG1, IgG2a, IgG2b and IgG3 in the case of mouse, andIgG1, IgG2, IgG3 and IgG4 in the case of human. The mouse IgG1 and IgG2aand human IgG1 types have complement-dependent cytotoxicity (hereinafterreferred to as “CDC”) and antibody-dependent cellular cytotoxicity(hereinafter referred to as “ADCC”), so that they are useful in applyingto medical treatments.

2. Production of Recombinant Antibody (I)—Anti-human VEGF Receptor Flt-1Humanized Antibody

(1) Construction of Humanized Antibody Expression Vector

In order to produce a humanized antibody from a non-human animalantibody, a humanized antibody expression vector is prepared. Thehumanized antibody expression vector is a vector for expression inanimal cells into which a gene encoding CH and CL, C regions of a humanantibody, have been inserted. Such an expression vector is constructedby inserting two genes, one encoding CH of a human antibody and theother encoding CL of a human antibody, into an expression vector foranimal cells.

Any C regions of a human antibody such as Cγ1 and Cγ4 of a humanantibody H chain, Cκ of a human antibody L chain and the like can beused. A chromosomal DNA consisting of an exon(s) and an intron(s) orcDNA can be used as a gene encoding a C region of a human antibody. Anyexpression vectors can be used as expression vectors for animal cells,provided that they can incorporate and express a gene encoding a Cregion of a human antibody.

Examples include pAGE107 [Cytotechnology, 3: 133 (1990)], PAGE103 [J.Biochem., 101: 1307 (1987)], pHSG274 [Gene, 27: 223 (1984)], pKCR [Proc.Natl. Acad. Sci. USA, 78: 1527 (1981)] and pSGI βd2-4 [Cytotechnology,4: 173 (1990)]. A promoter and an enhancer to be used in preparation ofan expression vector for animal cells are exemplified by an SV40 earlypromoter and enhancer [J. Biochem., 101: 1307 (1987)], a Moloney mouseleukemia virus LTR promoter and enhancer [Biochem. Biophys. Res. Comun.,149: 960 (1987)], an immunoglobulin H chain promoter [Cell, 41: 479(1985)] and enhancer [Cell, 33: 717 (1983)], and the like.

The humanized antibody expression vector may be either of a type inwhich a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In terms of ease ofconstruction of a humanized antibody expression vector, easiness ofintroduction into animal cells, balance between the expression amountsof antibody H and L chains in the animal cells and for other reasons, atandem type of humanized antibody expression vector is more preferred[J. Immunol. Methods, 167: 271 (1994)].

(2) Preparation of cDNA Encoding VH and VL of Non-human animal Antibody

cDNA encoding VH and VL of a non-human animal antibody such as a mouseanti-human VEGF receptor Flt-1 monoclonal antibody is obtained, forexample, as follows:

mRNA is extracted from an anti-human VEGF receptor Flt-1 monoclonalantibody-producing cell such as a mouse anti-human VEGF receptor Flt-1antibody-producing hybridoma and used to synthesize cDNA. Thesynthesized cDNA is inserted into a vector such as a phage or a plasmidto prepare a cDNA library. From the library, with a portion in a V or Cregion of a non-human animal antibody such as a mouse antibody beingused as a probe, a recombinant phage or plasmid which contains cDNAencoding VH and a recombinant phage or plasmid which contains cDNAencoding VL are isolated separately. The full nucleotide sequences of VHand VL of an antibody of interest which exist on the recombinant phageor plasmid are determined and the full amino acid sequences of the VHand VL are deduced from the nucleotide sequences.

(3) Construction of Human Chimeric Antibody Expression Vector

A human chimeric antibody expression vector can be constructed byinserting cDNA encoding VH and VL of a non-human animal antibody in aregion upstream of the gene encoding CH and CL of the human antibody onthe humanized antibody expression vector which has been constructed in2(1). For example, a restriction enzyme recognition site for cloning ofcDNA encoding VH and VL of a non-human animal antibody is createdpreliminarily in a region upstream of a gene encoding CH and CL of thehuman antibody on a chimeric antibody expression vector. At the cloningsite, cDNA encoding a V region of a non-human animal antibody isinserted through a synthetic DNA (see below) to prepare a human chimericantibody expression vector. The synthetic DNA consists of a nucleotidesequence at the 3′ end of a V region of the non-human animal and anucleotide sequence at the 5′ end of a C region of the human antibodyand are prepared by a DNA synthesizer such that it has appropriaterestriction enzyme sites at both ends.

(4) Identification of CDR Sequences of Non-human Animal antibody

VH and VL which form an antigen-binding site of an antibody consist of 4framework regions (hereinafter referred to as “FR regions”) havingrelatively conserved sequences and 3 complementarity-determining regions(CDRs) having a wide variety of sequences which link the FR regions[Sequences of Proteins of Immunological Interest, US Dept. Health andHuman Services (1991) (hereinafter referred to as “Sequences of Proteinsof Immunological Interest”]. The amino acid sequence of the respectiveCDR (CDR sequence) can be identified by comparison with the amino acidsequences of V regions of known antibodies (Sequences of Proteins ofImmunological Interest).

(5) Construction of cDNA Encoding V Region of Human CDR-grafted antibody

cDNA encoding VH and VL of a human CDR-grafted antibody can be obtainedas follows:

In the first step, for each of VH and VL, the amino acid sequence of FRin a V region of a human antibody to which CDR in a V region of anon-human animal antibody of interest is to be grafted is selected. Anyamino acid sequences of FRs in V regions derived from human antibodiescan be used as the amino acid sequences of FRs in V regions of humanantibodies.

For example, the amino acid sequences of FRs in V regions of humanantibodies recorded in Protein Data Bank and amino acid sequences commonto subgroups of FRs in V regions of human antibodies (Sequences ofProteins of Immunological Interest) can be used. In order to produce ahuman CDR-grafted antibody having an excellent activity, an amino acidsequence having high homology, preferably homology of 65% or more, withthe amino acid sequence of a V region of a non-human animal antibody ofinterest is desired. In the second step, a DNA sequence encoding theselected amino acid sequence of FR in a V region of a human antibody isligated to a DNA sequence encoding the amino acid sequence of CDR in a Vregion of a non-human animal antibody and a DNA sequence encoding theamino acid sequences of VH and VL is desired. In order to obtain a DNAsequence designed to construct a CDR-grafted antibody variable regiongene, several synthetic DNAs are designed for each chain such that thefull DNA sequence is covered. Using the synthetic DNAs, polymerase chainreaction (hereinafter referred to as “PCR”) is performed. For eachchain, preferably 6 synthetic DNAs are designed in view of the reactionefficiency of PCR and the lengths of DNAs which can be synthesized.After the reaction, amplified fragments are subcloned into appropriatevectors and their nucleotide sequences are determined to -obtain aplasmid which contains cDNA encoding the amino acid sequence of a Vregion of each chain of a human CDR-grafted antibody of interest.Alternatively, cDNA encoding the amino sequence of a V region of eachchain of a human CDR-grafted antibody of interest may be constructed bysynthesizing the full sequences of sense and antisense strand usingsynthetic DNAs consisting of about 100 bases and annealing and ligatingthem.

(6) Modification of the Amino Acid Sequence of V Region of HumanCDR-grafted Antibody

It is known that if a human CDR-grafted antibody is prepared by simplygrafting only CDR in a V region of a non-human animal antibody ofinterest between FRs in a V region of a human antibody, its activity islower than that of the original non-human animal antibody[BIO/TECHNOLOGY, 9: 266 (1991)]. Hence, among the amino acid sequencesof FR in a V region of a human antibody, an amino acid residue whichtakes part in direct binding to an antigen, an amino acid residue whichinteracts with an amino acid residue in CDR, or an amino acid residuewhich may take part in the maintenance of the steric structure of anantibody is modified to an amino acid residue that is found in theoriginal non-human animal antibody such that the activity of the humanCDR-grafted antibody is increased. For efficient identification of theamino acid residue, the steric structure of an antibody is constructedand analyzed by X-ray crystallography, computer-modeling or the like.However, no method for producing a human CDR-grafted antibody which canbe applied to any antibodies has yet been established and, therefore,various attempts must currently be made on a case-by-case basis.

The modification of the selected amino acid sequence of FR in a V regionof a human antibody can be accomplished using various primers formutation by PCR described in 2(5). Amplified fragments obtained by thePCR are subcloned into appropriate vectors and their nucleotidesequences are determined to obtain a vector containing cDNA into which amutation of interest has been introduced (hereinafter referred to as“amino acid sequence-replaced vector”).

Alternatively, the modification of an amino acid sequence in a narrowregion may be accomplished by a PCR-mutagenesis method using primers formutation consisting of 20-35 bases. More specifically, a sense mutationprimer and an antisense mutation primer which consist of 20 to 35 basesand which contain DNA sequences encoding the amino acid residue to bemodified are synthesized and used to perform 2-step PCR using as aplasmid as a template which contains cDNA encoding the amino acidsequence of a V region which is to be modified. The finally amplifiedfragments are subcloned into appropriate vectors and their nucleotidesequences are determined to obtain an amino acid sequence-modifiedvector containing cDNA into which a mutation of interest has beenintroduced.

(7) Construction of Human CDR-grafted Antibody Expression Vector

A human CDR-grafted antibody expression vector can be constructed byinserting the cDNA encoding VH and VL of the human CDR-grafted antibodyobtained in 2(5) and 2(6) in a region upstream of the gene encoding CHand CL of the human antibody in the humanized antibody expression vectordescribed in 2(1). For example, if recognition sites for appropriateenzymes are introduced at the ends of the 5′ and 3′ terminal syntheticDNAs during PCR for construction of cDNA encoding the amino acidsequences of VH and VL of the human CDR-grafted antibody, the cDNA canbe inserted in a region upstream of a gene encoding a C region of adesired human antibody such that it is expressed in an appropriate form.

(8) Transient Expression of Humanized Antibodies and Evaluation of theirActivities

In order to evaluate the activities of a wide variety of humanizedantibodies efficiently, the human chimeric antibody expression vectordescribed in 2(3), and the human CDR-grafted antibody expression vectordescribed in 2(7) or their modified vectors may be transfected intoCOS-7 cells (ATCC CRL 1651) and humanized antibodies expressedtransiently [Methods in Nucleic Acids Res., CRC Press, p. 283, (1991)],followed by determination of their activities.

Examples of the method for transfecting the expression vector into aCOS-7 cell include a DEAE-dextran method [Methods in Nucleic Acids Res.,CRC Press, p. 283, (1991)], a lipofection method [Proc. Natl. Acad. Sci.USA, 84: 7413 (1987)] and the like.

After transfection of the vector, the activities of the humanizedantibodies in the culture supernatant can be determined by the enzymeimmunoassay (ELISA) described in 1(5) and the like.

(9) Stable Expression of Humanized Antibodies and Evaluation of theirActivities

Transformants which produce a humanized antibody stably can be obtainedby transfecting into appropriate host cells the human chimeric antibodyexpression vector described in 2(3) and the human CDR-grafted antibodyexpression vector described in 2(7).

Examples of the method for transfecting the expression vector into hostcells include electroporation [Japanese Published Unexamined PatentApplication No. 257891/90, Cytotechnology, 3: 133 (1990)] and the like.

Any cells can be used as host cells into which the humanized antibodyexpression vector is to be transfected, provided that they can express ahumanized antibody. Examples include mouse SP2/O—Ag14 cell (ATCCCRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cells which aredetective in dihydrofolate reductase gene (hereinafter referred to as“DHFR gene”) [Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)] and ratYB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as“YB2/0 cell”).

After transfection of the vector, transformants which express ahumanized antibody stably are selected in accordance with the methoddisclosed in Japanese Published Unexamined Patent Application No.257891/90, using an RPMI1640 medium containing G418 and FCS. Thehumanized antibody can be produced and accumulated in a culture mediumby culturing the selected transformants in a medium. The activity of thehumanized antibody in the culture medium is determined by the methoddescribed in 1(5) or the like. The production of the humanized antibodyby the transformants can be increased by the method described inJapanese Published Unexamined Patent Application No. 257891/90,utilizing a DHFR gene-amplification system or the like.

The humanized antibody can be purified from the culture supernatant ofthe transformants by using a protein A column [Antibodies, A LaboratoryManual, Cold Spring harbor, Chapter 8 (1988)]. Any other conventionalmethods for protein purification can be used. For example, the humanizedantibody can be purified by a combination of gel filtration,ion-exchange chromatography, ultrafiltration and the like. The molecularweight of the H chain or L chain of the purified humanized antibody orthe antibody molecule as a whole is determined by polyacrylamide gelelectrophoresis (SDS-PAGE) [Nature, 227: 680, (1970)], Western blotting[Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter12 (1988)] and the like.

The reactivity of the purified humanized antibody and the inhibitionactivity of the humanized antibody against human VEGF receptor Flt-1 canbe determined by the method described in 1 (5).

3. Method of Use of Recombinant Antibody (II)

(1) Preparation Method of Antibody Fragments, Fab, Fab′ and F(ab′)₂

Antibody fragments are formed by treating the above antibody with anenzyme. Examples of the enzyme include papain and trypsin.

Alternatively, Fab, Fab′ or F(ab′)₂ can also be produced by inserting aDNA fragment which encodes the Fab, Fab′ or F(ab′)₂ fragment of theanti-human VEGF receptor Flt-1 antibody into an expression vector foranimal cells and introducing the vector into cells of an animal toexpress the fragment of interest.

The thus formed antibody fragments can be purified by carrying outsuitable combination of techniques such as gel filtration, ion exchangechromatography, affinity chromatography and ultrafiltration. Molecularweight of the thus purified Fab, Fab′ or F(ab′)₂ fragment is measured bypolyacrylamide gel electrophoresis (SDS-PAGE) [Nature, 227: 680 (1970)]or [Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory,Chapter 12 (1988)].

Reactivity of the thus purified Fab, Fab′ or F(ab′)₂ and bindingactivity of Fab, Fab′ or F(ab′)₂ upon VEGF receptor Flt-1 can bemeasured by the method described in the aforementioned step 1-(5) or thelike.

(2) Construction of Anti-human VEGF Receptor Flt-1 scFv

A vector for expression of scFv of a non-human animal antibody or scFvof a human CDR-grafted antibody can be constructed by inserting intoscFv expression vector the cDNAs encoding VH and VL of a non-humananimal antibody or a human CDR-grafted antibody which are described in2(2), 2(5) and 2(6). Any expression vectors can be used as scFvexpression vectors, provided that they can incorporate and express thecDNAs encoding VH and VL of a non-human animal antibody or a humanCDR-grafted antibody.

Examples include pAGE107 [Cytotechnology, 3: 133 (1990)], pAGE103 [J.Biochem., 101: 1307 (1987)]), pHSG274 [Gene, 27: 223 (1984)], pKCR[Proc. Natl. Acad. Sci. USA, 78: 1527 (1981)] and pSGI βd2-4[Cytotechnology, 4: 173 (1990)]. A host for expressing scFv can beselected from among E. coli, yeast, animal cells, and the like. In thiscase, an expression vector which is compatible with the specific hostshould be selected. The scFv can be secreted out of the cell andtransported into the periplasm region or retained within the cell byinserting a cDNA encoding an appropriate signal peptide into theexpression vector.

An scFv expression vector into which the cDNA encoding scFv of interesthas been inserted can be constructed by inserting the cDNA encoding scFvconsisting of VH-P-VL or VL-P-VH (where P is a peptide linker) into theselected expression vector in a region downstream of an appropriatepromoter and a signal peptide.

The cDNA encoding scFv can be obtained by linking a VH encoding cDNA toa VL encoding cDNA through a synthetic DNA encoding a peptide linkerhaving recognition sites for appropriate restriction enzymes at both theends. It is important to optimize the linker peptide such that itsaddition does not interfere with the binding of VH and VL to an antigen.For example, the linker descried by Pantoliano et al. [Biochemistry, 30:10117 (1991)] and its modified versions may be used.

(3) Production of Anti-human VEGF Receptor Flt-1 dsFv

dsFv can be produced by a process comprising the steps of providingcDNAs encoding VH and VL of a non-human animal antibody or cDNAsencoding VH and VL of a human CDR-grafted antibody, modifying the DNAsequence which corresponds to a one-amino acid residue at an appropriateposition in the respective cDNA with a DNA sequence corresponding to acysteine residue, expressing the modified cDNAs and purifying theresultant peptide and then forming a disulfide bond. The modification ofan amino acid residue to a cysteine residue can be performed by amutagenesis method using PCR described in 2 (5).

A dsFv H chain expression vector and a dsFv L chain expression vectorcan be constructed by inserting the resulting cDNAs encoding themodified VH and modified VL into appropriate expression vectors. Anyexpression vectors can be used as dsFv expression vectors, provided thatthey can incorporate and express cDNAs encoding a modified VH and amodified VL. For example, pAGE107 [Cytotechnology, 3: 133 (1990)],pAGE103 [J. Biochem., 101: 1307 (1987)], pHSG274 (Gene, 27: 223 (1984)],pKCR [Proc. Natl. Acad. Sci. USA, 78: 1527 (1981)], pSGI βd2-4[Cytotechnology, 4: 173 (1990)] and the like can be used. A host used toexpress a dsFv L chain expression vector and a dsFv H chain expressionvector for formation of dsFv can be selected from among E. coli, yeast,animal cells, and the like. In this case, an expression vector which iscompatible with the specific host should be selected. The dsFv can besecreted out of the cell and transported into the periplasm region orretained within the cell by inserting a cDNA encoding an appropriatesignal peptide into the expression vector.

(4) Expression of Various Antibodies and Evaluation of their Activity

A transformant which produces an antibody fragment, scFv, a dsFv H chainor a dsFv L chain of interest can be obtained by transfecting into ahost cell the antibody fragment expression vector, the scFv expressionvector, the dsFv H chain expression vector or the dsFv L chainexpression vector that was constructed in 3(1) to 3(3) byelectroporation [Japanese Published Unexamined Patent Application No.257891/90; Cytotechnology, 3: 133 (1990)] or the like. Afterintroduction of the expression vector, the expression of the antibodyfragment, scFv, dsFv H chain, or dsFv L chain in the culture supernatantor the like can be confirmed by the method described in 1(5).

The collection and purification of the antibody fragment, scFv, dsFv Hchain, or dsFv L chain can be accomplished by combinations of knowntechniques. For example, if the antibody fragment, scFv, dsfv H chain,or dsFv L chain is secreted in a medium, they can be concentrated byultrafiltration and their collection and purification can be thenperformed by various types of chromatography or gel filtration. If theantibody fragment, scFv, dsFv H chain, or dsFv L chains is transportedinto the periplasm region of the host cell, they can be concentrated byultrafiltration after the application of an osmotic shock to the celland their collection and purification can be then performed by varioustypes of chromatography or gel filtration. If the antibody fragment,scFv, dsFv H chain, or dsFv L chain is insoluble and exists as a granule(i.e., inclusion body), their collection and purification can beperformed by lysis of the cells, repeated centrifugation and washing forisolation of the granule, solubilization with guanidine-HCl andsubsequent performance of various types of chromatography or gelfiltration.

The purified scFv can be measured by the method described in the above1(5) and the like.

The purified dsFv H chain and dsFv L chain are mixed and subjected to arefolding procedure for deriving an active structure [MolecularImmunology, 32: 249 (1995)] to form a disulfide bond. Subsequently, theactive dsFv can be purified by antigen affinity chromatography orion-exchange chromatography or gel filtration. The activity of the dsFvcan be determined by the method described in 1(5) or the like.

4. Preparation Method of Fusion Antibody and Fusion Peptide

The fusion antibodies prepared by chemically or by gene-engineeringlylinking a radioactive isotope, a protein or a low molecular agent withthe antibody or antibody fragment of the present invention can also beused as derivatives of the antibody.

A fusion antibody in which an antibody and a toxic protein arechemically linked can be prepared in accordance, for example, with themethod described in Anticancer Research, 11: 2003 (1991) or NatureMedicine, 3: 350 (1996).

A fusion antibody in which an antibody and a toxin or a protein such ascytokine is gene-engineeringly linked can be prepared in accordance, forexample, with the method described in Proc. Natl. Acad. Sci. USA, 93:974 (1996) or Proc. Natl. Acad. Sci. USA, 93: 7826 (1996).

A fusion antibody in which an antibody and a low molecular weightanticancer agent are chemically linked can be prepared in accordance,for example, with the method described in Science, 261: 212 (1993).

A fusion antibody in which an antibody and a radioactive isotope arechemically linked can be prepared in accordance, for example, with themethod described in Antibody Immunoconjugates and Radiopharmaceuticals,3: 60 (1990) or Anticancer Research, 11: 2003 (1991).

Examples of the low molecular agent include antibacterial agents, suchas alkylating agents (e.g., nitrogen mustards, cyclophosphamide),antimetabolites (e.g., 5-fluorouracil, methotrexate), antibiotics (e.g.,mitomycin C, daunorubicin), plant alkaloids (e.g., vincristine,vinblastine), and hormone agents (e.g., tamoxifen, dexamethasone)[Clinic Oncology, edited by Japan Clinic Oncology Society, published byCancer and Chemotherapy Corporation (1996)]; and antiinflammatoryagents, such as steroid drugs (e.g., hydrocortisone, prednisone),non-steroid drugs (e.g., aspirin, indomethacin), immunomodulators (e.g.,cyclophosphamide, azathioprine), and antihistamic agents (e.g.,chlorpheniramine maleate, clemastine) [Inflammation andAnti-Inflammatory Therapy, published by Ishiyaku Shuppan (1982)].

It is expected that these derivatives can provide more effective andside effect-reduced diagnoses and treatments by accumulating aradioactive isotope, a protein (such as cytokine, a toxin or an enzyme)or a low molecular agent within the peripheral of a target tissue, basedon the specificity of antibody molecules.

The fusion peptide of the present invention can be prepared in the samemanner as the fusion antibody of the present invention described above.

5. Method of Use of Antibody and Peptide (I)

The antibodies and peptides of the present invention specific for humanVEGF receptor Flt-1 can be used as a diagnostic agent or a treatingagent of diseases in which the morbid states progress abnormalangiogenesis, which include proliferation or metastasis of solid tumorssuch as breast cancer, prostatic cancer, large bowel cancer, gastriccancer and lung cancer, arthritis in chronic rheumatoid arthritis,diabetic retinopathy, retinopathy of prematurity and psoriasis.

In addition, since the antibodies and peptides of the present inventioncan inhibit biological activities of human VEGF, auto-phosphorylation ofFlt-1 can be inhibited by inhibiting binding of human VEGF to Flt-1. Asa result, they can inhibit VEGF-dependent proliferation of humanvascular endothelial cells and therefore can be used as an agent fortreating diseases in which their morbid states progress by abnormalangiogenesis, which include proliferation or metastasis of solid tumorssuch as breast cancer, prostatic cancer, large bowel cancer, gastriccancer and lung cancer, arthritis in chronic rheumatoid arthritis,diabetic retinopathy, retinopathy of prematurity and psoriasis.

The above-described anti-human VEGF receptor Flt-1 antibodies, peptides,and fusion antibodies and fusion peptides thereof fused with othermolecules are linked to human VEGF receptor Flt-1 and, via antibodyeffector activities such as ADCC and CDC, destroy cells in which VEGFreceptor Flt-1 is expressed on the cell surface, so that they are usefulin treating diseases in which the morbid states progress by abnormalangiogenesis, which include proliferation or metastasis of solid tumors,such as breast cancer, prostatic cancer, large bowel cancer, gastriccancer and lung cancer, arthritis in chronic rheumatoid arthritis,diabetic retinopathy, retinopathy of prematurity and psoriasis.

The pharmaceutical preparation containing the antibody or peptide of thepresent invention can be administered directly as a treating agent, butit is generally preferred to provide it in the form of a pharmaceuticalmedicament produced by mixing it with at least one pharmacologicallyacceptable carrier in accordance with optional methods well known in thetechnical field of pharmaceutics.

It is preferred to select a route of administration which is the mosteffective in carrying out the intended treatment, such as oraladministration or parenteral administration that includes trachealadministration, rectal administration, subcutaneous injection,intramuscular injection and intravenous injection. Intravenous injectionis preferred in the case of an antibody or peptide preparation.

The dosage form includes sprays, capsules, tablets, granules, syrups,emulsions, suppositories, injections, ointments, and tapes.

Examples of the pharmaceutical preparation suitable for oraladministration include emulsions, syrups, capsules, tablets, powders,and granules.

Liquid preparations, such as emulsions and syrups, are produced usingadditives, such as water, saccharides (e.g., sucrose, sorbitol,fructose), glycols (e.g., polyethylene glycol, propylene glycol), oils(e.g., sesame oil, olive oil, soybean oil), antiseptics (e.g.,p-hydroxybenzoic acid esters), and flavors (e.g., strawberry flavor,peppermint).

Solid preparations, such as capsules, tablets, powders and granules, canbe produced using additives, such as fillers (e.g., lactose, glucose,sucrose, mannitol), disintegrating agents (e.g., starch, sodiumalginate), lubricating agents (e.g., magnesium stearate, talc), binders(e.g., polyvinyl alcohol, hydroxypropylcellulose, gelatin), surfactants(e.g., fatty acid esters), and plasticizers (e.g., glycerol).

Preferred examples of pharmaceutical preparations suitable forparenteral administration include injections, suppositories and sprays.

Injections are prepared using a carrier, such as a salt solution,glucose solution or a mixture thereof.

Suppositories are prepared using a carrier such as cacao butter,hydrogenated fat or a carboxylic acid.

Sprays are prepared from the compound itself or using a carrier whichdoes not stimulate oral and airway mucous membranes of patients and canfacilitate absorption of the compound by dispersing it as minuteparticles.

Examples of the carrier include lactose and glycerol. Depending on theproperties of the antibody or peptide and the carrier to be used, otherpreparations, such as aerosols and dry powders, can be produced. Thecomponents exemplified as additives of oral preparations can also beadded to these parenteral preparations.

The dose and frequency of administration vary depending on theconditions such as intended therapeutic effect, administration method,treating period, age and body weight, but the dose is generally from 10μg/kg to 8 mg/kg per day per adult.

The methods for the examination of antitumor effects of the antibody orpeptide of the present invention upon various tumor cells includecomplement-dependent cytotoxicity (CDC) activity assay, andantibody-dependent cellular cytotoxicity (ADCC) activity assay as invitro tests. An antitumor test in which a tumor system of anexperimental animal, such as mouse, is used can be exemplified as invivo tests.

The CDC activity assay, the ADCC activity assay and the antitumor testcan be carried out in accordance, for example, with the methodsdescribed in the literature [Cancer Immunology Immunotherapy, 36: 373(1993); Cancer Research, 54: 1511 (1994)].

Furthermore, the present invention relates to, using the antibody orpeptide of the present invention, a method for immunologically detectinghuman VEGF receptor Flt-1 or cells in which human VEGF receptor Flt-1 isexpressed on the surface thereof, a method for immunologically detectingand determining soluble human VEGF receptor Flt-1, and a method forinhibiting binding of a human VEGF to human VEGF receptor Flt-1 orbiological activities of human VEGF.

Moreover, the present invention relates to a diagnostic agent fordiseases in which their morbid states progress by abnormal angiogenesis,such as proliferation or metastasis of solid tumors, arthritis inrheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity,psoriasis, and the like.

The methods for detecting and determining human VEGF receptor Flt-1 aredescribed below.

6. Method of Use of Antibody or Peptide (II)

The method for detecting a disease in which the morbid states progressby abnormal angiogenesis includes a fluorescent antibody method, anenzyme-linked immunosorbent assay (ELISA), a radioactive materiallabeled immunoassay (RIA), an immunocyte staining method, animmunotissue staining method, Western blotting method, animmunoprecipitation method, and the like.

The fluorescent antibody method comprises the steps of: (a) separatinghuman cell or a crushing solution thereof, tissue or a crushing solutionthereof, serum, preural fluid, ascites fluid, ocular fluid or the liketo prepare a sample; (b) reacting the separated sample prepared in thestep (a) with the antibody or peptide of the present invention; (c)further reacting the reacted sample prepared in the step (b) with ananti-mouse IgG antibody or binding fragment labeled with a fluorescencesubstance, such as fluorescin isothiocyanate (FITC) or the like; and (d)measuring the fluorescence substance with a flow cytometer.

The enzyme-linked immunosorbent assay (ELISA) comprises the steps of:(a) separating human cell or a crushing solution thereof, tissue or acrushing solution thereof, serum, preural fluid, ascites fluid, ocularfluid or the like to prepare a sample; (b) reacting the separated sampleprepared in the step (a) with the antibody or peptide of the presentinvention; (c) further reacting the reacted sample prepared in the step(b) with an anti-mouse IgG antibody or binding fragment labeled with anenzyme, such as peroxydase, biotin or the like; and (d) measuring theresultant developed dye with an absorption measuring apparatus.

The radioactive material labeled immunoassay (RIA) comprises the stepsof: (a) separating human cell or a crushing solution thereof, tissue ora crushing solution thereof, serum, preural fluid, ascites fluid, ocularfluid, or the like to prepare a sample; (b) reacting the separatedsample prepared in the step (a) with the antibody or peptide of thepresent invention; (c) further reacting the reacted sample prepared inthe step (b) with an anti-mouse IgG antibody or binding fragment labeledwith radioactive ray; and (d) measuring the radioactive ray with ascintillation counter or the like.

The immunocyte staining and immunotissue staining methods comprise thesteps of: (a) separating human cell, tissue or the like to prepare asample; (b) reacting the separated sample prepared in the step (a) withthe antibody or peptide of the present invention; (c) further reactingthe reacted sample prepared in the step (b) with an anti-mouse IgGantibody or binding fragment labeled with a fluorescence substance, suchas fluorescin isothiocyanate (FITC) or the like, or an enzyme, such asperoxydase, biotin, or the like; and (d) observing the cell, tissue orthe like with a microscope.

Examples of the methods, using the monoclonal antibody of the presentinvention, for immunologically detecting human VEGF receptor Flt-1 or acell in which human VEGF receptor Flt-1 is expressed on the surfacethereof and for immunologically detecting and determining soluble humanVEGF receptor Flt-1 include immunocyte staining, Western blotting,sandwich ELISA, and the like. These methods are described below.

(1) Immunocyte Staining Using Monoclonal Antibody

First, the cells in which human VEGF receptor Flt-1 is expressed on thecell surface are prepared. Suspending cells as such or adherent cellsafter detachment of the cells using trypsin-EDTA are suspended, forexample, in a buffer solution for immunocyte staining use (PBScontaining 1% BSA, 0.02% EDTA and 0.05% sodium azide) and dispensed in1×10⁵ to 2×10⁶ portions. A culture supernatant of the anti-human VEGFreceptor Flt-1 monoclonal antibody-producing hybridoma obtained in theabove-described step 1-(4), the purified monoclonal antibody obtained inthe above-described step 1-(6) or the monoclonal antibody labeled withbiotin by a known method [Enzyme Antibody Method: published by GakusaiKikaku (1985)] is diluted with the buffer solution for immunocytestaining use or the buffer solution for immunocyte staining use furthersupplemented with 10% animal serum to a concentration of 0.1 to 50 μg/mland dispensed in 20 to 500 μl portions to carry out the reaction undercooling for 30 minutes. When the culture supernatant of the anti-humanVEGF receptor Flt-1 monoclonal antibody-producing hybridoma obtained inthe above-described step 1-(4) or the purified monoclonal antibodyobtained in the above-described step 1-(6) is used in the reaction, thecells are washed with the buffer solution for immunocyte staining useand then an anti-mouse immunoglobulin antibody or anti-ratimmunoglobulin labeled with fluorescence dye, such as FITC,phycoerythrin, or the like, which is dissolved in the buffer solutionfor immunocyte staining use to a concentration of about 0.1 to 50 μg/ml,is dispensed in 50 to 500 μl portions to carry out the reaction undercooling for 30 minutes. When the monoclonal antibody labeled with biotinis used in the reaction, streptoavidin is dispensed in 50 to 500 μlportions and then the reaction is carried out under cooling in the darkfor 30 minutes. After completion of the reaction, the cells arethoroughly washed with the buffer solution for immunocyte staining useand analyzed by a cell sorter.

(2) Detection of Human VEGF Receptor Flt-1 by Western Blotting

Cell membrane components are prepared from cells in which human VEGFreceptor Flt-1 is expressed, such as human. VEGF receptorFlt-1-expressing NIH3T3 cells (referred to as “NIH3T3-Flt-1”hereinafter), and from control cells such as NIH3T3 cells (referred toas “NIH3T3-Neo” hereinafter) [Oncogene, 10: 135 (1995)], and themembrane components are electrophoresed by the SDS-PAGE method underreducing conditions in an amount of 0.1 to 30 μl as protein per lane.The thus treated proteins are transferred on a PVDF membrane and allowedto react with PBS containing 1% BSA at room temperature for 30 minutesto carry out blocking. They are allowed to react with the culturesupernatant of the anti-human VEGF receptor Flt-1 monoclonalantibody-producing hybridoma obtained in the above-described step 1-(4)or the purified monoclonal antibody obtained in the above-described step1-(6), washed with PBS containing 0.05% Tween and then allowed to reactwith peroxidase-labeled goat anti-mouse IgG at room temperature for 2hours. After washing with PBS containing 0.05% Tween, bands to which theanti-human VEGF receptor Flt-1 monoclonal antibody is linked aredetected using ECL™ Western blotting detection reagents (manufactured byAmersham) or the like.

(3) Determination of Soluble VEGF Receptor Flt-1 Using MonoclonalAntibody

As a first antibody, the purified monoclonal antibody obtained in theabove-described step 1-(6) is coated on an appropriate plate and allowedto react with 0.056 to 10,000 ng/ml of the purified soluble anti-humanVEGF receptor Flt-1 monoclonal antibody-producing hybridoma obtained inthe above-described step 1-(1), or with a sample, such as human serum orthe like. After through washing of the plate, this is allowed to reactwith a second antibody, namely a monoclonal antibody labeled withbiotin, an enzyme, a chemiluminescence substance, a radioactive compoundor the like, which is one of the purified monoclonal antibodies obtainedin the above-described step 1-(6) but recognizes different epitope fromthat of the monoclonal antibody used as the first antibody, underreaction conditions suitable for binding of the labeled antibody to theepitope with which it binds. A calibration curve is prepared based onthe reactivity for the purified soluble VEGF receptor Flt-1, and theconcentration of the soluble VEGF receptor Flt-1 in the samples iscalculated.

The activity of inhibiting binding of human VEGF and human VEGF receptorFlt-1 can be confirmed by carrying out a VEGF-VEGF receptor Flt-1binding inhibition test using the antibody of the present invention inaccordance, for example, with the measuring method of binding betweengrowth factors and receptors described in New Biochemical ExperimentCourse, Vol. 7, Growth and Differentiation Factors and their Receptors,published by Tokyo Kagaku Dojin (1991).

In this method, for example, VEGF labeled with a radioactive material isallowed to react with cells or tissues in which Flt-1 is expressed, andthe VEGF linked to the Flt-1 expressing cells or tissues is measuredusing a scintillation counter. The activity of inhibiting binding ofVEGF labeled with a radioactive material or the like to Flt-1 can bemeasured by reacting the antibody or peptide of the present inventiontogether with the radioactive material-labeled VEGF.

Auto-phosphorylation inhibition activity of VEGF receptor Flt-1 can beconfirmed by carrying out a VEGF-VEGF receptor Flt-1auto-phosphorylation inhibition test using a antibody or a peptide inaccordance, for example, with the measuring method ofauto-phosphorylation of growth factor receptors described in SecondSeries Biochemical Experiment Course, Signal Transduction and CellularResponse, published by Tokyo Kagaku Dojin (1986).

In this method, VEGF is allowed to react with cells or tissues in whichFlt-1 is expressed, and auto-phosphorylation of Flt-1 accelerated by thebinding of VEGF is detected by immunoprecipitation or Western blotting.The activity of inhibiting auto-phosphorylation of Flt-1 accelerated bythe binding of VEGF can be measured by reacting the antibody or peptideof the present invention together with VEGF.

The activity of inhibiting biological activities of human VEGF can beconfirmed by carrying out growth, migration and tube formation tests ofVEGF dependent vascular endothelial cells [New Biochemical ExperimentCourse, Vol. 10, Blood Vessels (Endothelium and Smooth Muscle),published by Tokyo Kagaku Dojin (1991)].

The growth test of VEGF dependent vascular endothelial cells is a methodin which VEGF is allowed to react with vascular endothelial cells, andthe growth enhancing activity of vascular endothelial cells acceleratedby the binding of VEGF is measured by counting the number of cells. Theactivity of inhibiting growth enhancing activity of vascular endothelialcells accelerated by VEGF can be measured by reacting the antibody orpeptide of the present invention together with VEGF.

The migration test of VEGF dependent vascular endothelial cells is amethod in which VEGF is allowed to react with vascular endothelialcells, and the wondering enhancing activity of vascular endothelialcells accelerated by the binding of VEGF is observed under a microscope.The activity of inhibiting aberrance enhancing activity of vascularendothelial cells accelerated by VEGF can be measured by reacting theantibody or peptide of the present invention together with VEGF.

The tube formation test of VEGF dependent vascular endothelial cells isa method in which VEGF is allowed to react with vascular endothelialcells, and the tube formation enhancing activity of vascular endothelialcells accelerated by the binding of VEGF is observed under a microscope.The activity of inhibiting tube formation enhancing activity of vascularendothelial cells accelerated by VEGF can be measured by reacting theantibody or peptide of the present invention together with VEGF.

The inhibition methods of the bonding of human VEGF to human VEGFreceptor Flt-1 and of the biological activities of human VEGF areexemplified.

(4) Inhibition Test of Binding of VEGF to VEGF Receptor Flt-1 UsingMonoclonal Antibody

Methanol is dispensed in 100 μl portions into wells of a 96 wellMultiScreen-IP Plate (manufactured by Millipore) to give hydrophilicnature to the PVDF membrane on the bottom of the plate. After washingwith water, human VEGF receptor Flt-1 or a recombinant protein such as afusion protein with human VEGF receptor Flt-1 is diluted to aconcentration of 0.1 to 10 μg/ml, dispensed in 50 μl/well portions andthen allowed to stand overnight at 4° C. for its adsorption. Afterwashing, PBS containing 1% bovine serum albumin (BSA) is dispensed in100 μl/well portions and the reaction is carried out at room temperaturefor 1 hour to block any remaining active groups. After washing with PBS,the culture supernatant of the anti-human VEGF receptor Flt-1 monoclonalantibody-producing hybridoma obtained in the above-described step 1-(4)or the purified monoclonal antibody obtained in the above-described step1-(6) is dispensed in 50 μl/well portions and then 0.1 to 10 ng/ml of¹²⁵I-labeled VEGF (manufactured by Amersham) is dispensed in 50 μl/wellportions, subsequently carrying out the reaction at room temperature for1.5 hours. After washing with 0.05% Tween-PBS, the wells are dried at50° C., and a scintillator is dispensed in 20 to 100 μl/well portions tomeasure the radioactivity of the ¹²⁵I-labeled VEGF linked to each wellusing Top Count (manufactured by Packard) or the like.

(5) Inhibition Test of Binding of VEGF to VEGF Receptor Flt-1-ExpressingCells Using Monoclonal Antibody

PBS containing 1% bovine serum albumin (BSA) is dispensed in 100 μlportions into wells of a 96 well MultiScreen-HV Plate (manufactured byMillipore), the reaction is carried out at room temperature for 1 hourto block the active groups in wells and then NIH3T3-Flt-1 cellssuspended in 1% BSA-PBS containing 0.05% NaN₃ is dispensed in 1×10⁴ to1×10⁵/well portions. After washing with 1% BSA-PBS, the culturesupernatant of the anti-human VEGF receptor Flt-1 monoclonalantibody-producing hybridoma obtained in the above-described step 1-(4)or the purified monoclonal antibody obtained in the above-described step1-(6) is dispensed in 50 μl/well portions and then 0.1 to 10 ng/ml of¹²⁵I-labeled VEGF (manufactured by Amersham) is dispensed in 50 μl/wellportions, subsequently carrying out the reaction at room temperature for1.5 hours. After washing with PBS, the wells are dried at 50° C., and ascintillator is dispensed in 20 to 100 μl/well portions to measure theradioactivity of the ¹²⁵I-labeled VEGF linked to each well using TopCount (manufactured by Packard) or the like.

The other antibodies and peptides of the present invention can be usedin the same manner as the monoclonal antibody of the present inventiondescribed above.

The present invention renders possible provision of antibodies andpeptides that specifically binds to human VEGF receptor Flt-1 which isconsidered to be expressed specifically in vascular endothelial cells ofhuman angiogenesis regions. The antibodies and peptides of the presentinvention are useful for the immunological detection of humanangiogenesis regions by immunocyte staining and for the diagnosis ortreatment, through the inhibition of the biological activities of humanVEGF, of diseases in which their morbid states progress by abnormalangiogenesis, such as proliferation or metastasis of solid tumors,arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy ofprematurity, psoriasis, and the like.

The present invention will be explained by Examples below; however, theinvention is not limited thereto.

EXAMPLE 1

1. Preparation of Antigen.

(1) Construction of Soluble Human VEGF Receptor Flt-1 3N ExpressionVector

A vector was prepared in the following manner, for use in the expressionof a soluble human VEGF receptor Flt-1 fragment (hereinafter referred toas “soluble human VEGF receptor Flt-1 3N”) which corresponds to a regionof the 1st to 338th positions (including a signal sequence) from theN-terminal amino acid of human VEGF receptor Flt-1. The soluble humanVEGF receptor Flt-1 3N corresponds to the N-terminal side threeimmunoglobulin-like regions of the extracellular domain of the solublehuman VEGF receptor Flt-1.

A cDNA clone flt#3-7 [M. Shibuya et al., Oncogene, 5: 519 (1990)] whichcontains whole length cDNA encoding the human VEGF receptor Flt-1 waspartially digested with restriction enzymes EcoRI and TaqI to collect a1,263 bp EcoRI-TaqI DNA fragment from the 5′-end, and the thus collectedfragment was inserted into the 5′ side EcoRI site and 3′ side NotI sitedownstream of the transcription initiation point of the polyhedrin geneof a baculovirus gene recombinant vector pVL1393 plasmid (manufacturedby Invitrogen) using a TaqI-NotI adapter into which a termination codonhad been artificially introduced (a synthetic DNA fragment having thenucleotide sequences shown in the SEQ ID NO:1 and NO:2) to obtainsoluble human VEGF receptor Flt-1 3N expression vector pVL1393/Flt 3N(FIG. 1).

(2) Construction of Soluble Human VEGF Receptor Flt-1 7N ExpressionVector

A vector was prepared in the following manner, for use in the expressionof a soluble human VEGF receptor Flt-1 fragment (referred to as “solublehuman VEGF receptor Flt-1 7N” hereinafter) which corresponds to a regionof the 1st to 750th positions (including a signal sequence) from theN-terminal amino acid of human VEGF receptor Flt-1. The soluble humanVEGF receptor Flt-1 7N corresponds to the seven immunoglobulin-likeregions of the extracellular domain of the soluble human VEGF receptorFlt-1.

A 2.5 unit portion of Taq polymerase was added to 100 μl of 0.001% (w/v)gelatin solution of 10 mM MgCl₂ containing 10 pmol of primers having thenucleotide sequences shown in SEQ ID NO:3 and NO:4, 10 ng of flt#3-7clone [Oncogene, 5: 519, (1990)] DNA and 10 mM deoxynucleotidetriphosphates. The polymerase chain reaction (PCR) was repeated 30 timesin which one reaction consisted, after pretreatment at 95° C. for 5minutes, of treatments at 95° C. for 90 seconds, at 50° C. for 90seconds and finally at 72° C. for 90 seconds, subsequently collecting aDNA fragment. The DNA fragment was digested with HindIII (the 1893 bpposition in the flt#3-7 clone) and NotI to obtain a 610 bp HindIII-NotIDNA fragment, namely a DNA fragment containing a 1894-2499 bp fragmentof the flt#3-7 clone, termination codon and NotI recognition sequence.Next, the flt#3-7 clone was digested with restriction enzymes EcoRI andHindIII to collect an EcoRI-HindIII fragment of 1893 bp from the 5′-end.The 610 bp HindIII-NotI DNA fragment and the 1893 bp EcoRI-HindIIIfragments were then inserted into the 5′ side EcoRI site and 3′ sideNotI site downstream of the transcription initiation point of thepolyhedrin gene of a baculovirus gene recombinant vector pVL1393plasmid, thereby preparing soluble human VEGF receptor Flt-1 7Nexpression vector pVL1393/Flt 7N (FIG. 2).

(3) Preparation of Recombinant Virus for Use in the Expression ofSoluble Human VEGF Receptor Flt-1 in Insect Cells

For the production of protein by insect cells, it is necessary toprepare a recombinant virus into which a gene of interest is integrated,and the preparation process consists of a step in which a cDNA moleculeencoding a protein of interest is inserted into a special plasmid, whichis called a transfer vector, and a subsequent step in which a wild typevirus and the transfer vector are co-transfected into insect cells toobtain a recombinant virus by homologous recombination. These steps werecarried out in the following manner using BaculoGold Starter Kitmanufactured by Pharmigen (product no. PM-21001K) in accordance with themanual.

A recombinant baculovirus was prepared in the following manner byintroducing a filamentous baculovirus DNA (BaculoGold baculovirus DNA,produced by Pharmigen) and the thus prepared transfer vector DNA intoinsect cells Sf9 (manufactured by Pharmigen) which had been culturedusing TMN-FH insect medium (manufactured by Pharmigen), using alipofectin method [Protein, Nucleic Acid, Enzyme, 37: 2701 (1992)].

A 1 μg portion of pVL1393/Flt7N prepared in the above step (2) orpVL1393/Flt3N prepared in the above step (1) and 20 ng of filamentousbaculovirus DNA were dissolved in 12 μl of distilled water, the solutionwas mixed with a mixture of 6 μl lipofectin and 6 μl distilled water andthen the resulting mixture was allowed to stand at room temperature for15 minutes. Separately from this, 1×10⁶ of Sf9 cells were suspended in 2ml of Sf900-II medium (manufactured by Gibco) and put into a cellculture plastic Petri dish of 35 mm in diameter. To this was added wholevolume of the just described solution of plasmid DNA, filamentousbaculovirus DNA and lipofectin mixture, followed by 3 days of culturingat 27° C. to collect 1 ml of the culture supernatant containing therecombinant virus. A 1 ml portion of Sf900-II medium was added to theresulting Petri dish and 3 days of culturing was carried out at 27° C.to obtain an additional 1.5 ml of the recombinant virus containingculture supernatant.

Next, the thus obtained recombinant virus for use in the proteinexpression was grown in the following manner.

A 2×10⁷ portion of Sf9 cells were suspended in 10 ml of Sf900-II medium,put into a 175 cm² flask (manufactured by Greiner) and allowed to standat room temperature for 1 hour to adhere the cells to the flask. Thesupernatant fluid was subsequently discarded and 15 ml of fresh TMN-FEinsect medium and a 1 ml portion of the recombinant virus containingculture supernatant described above were added and cultured for 3 daysat 27° C. After the culturing, the supernatant fluid was centrifuged at1,500×g for 10 minutes to remove the cells to obtain a recombinant virussolution for use in the protein expression.

The titer of virus in the thus obtained recombinant virus solution wascalculated by the method described in BaculoGold Starter Kit Manual(Pharmigen).

A 6×10⁶ portion of Sf9 cells were suspended in 4 ml of Sf900-II medium,put into a cell culture plastic Petri dishes of 60 mm in diameter andallowed to stand at room temperature for 1 hour to adhere the cells tothe dish. Next, the supernatant fluid was discarded, 400 μl of freshSf900-II medium and the above-described recombinant virus solutiondiluted 10,000 times with Sf900-II medium were added to the dish andallowed to stand at room temperature for 1 hour, the medium was removedand then 5 ml of a medium containing 1% low melting point agarose(Agarplaque Agarose, produced by Pharmigen) (prepared by mixing 1 ml ofsterilized 5% Agarplaque plus agarose aqueous solution with 4 ml ofTMN-FH insect medium and stored at 42° C.) was poured into the dish.After standing at room temperature for 15 minutes, the dish was tiedwith a vinyl tape to prevent drying, put into a sealable plasticcontainer and then cultured at 27° C. for 6 days. A 1 ml portion of PBScontaining 0.01% of Neutral Red was added to the dish to carry out theadditional culturing for 1 day and then the number of the thus formedplaques was counted. By the above procedure, it was found that each ofthe recombinant virus solutions contained virus particles of about 1×10⁷plaque forming units (hereinafter referred to as “PFU”) per ml.

(4) Expression of Soluble Human VEGF Receptors Flt-1 7N and Flt-1 3N inInsect Cells and Purification Thereof

Soluble human VEGF receptors Flt-1 7N and Flt-1 3N were obtained in thefollowing manner. A 4×10⁷ portion of High Five cells were suspended in30 ml of EX-CELL™ 400 medium (manufactured by JRH Biosciences) containedin a 175 cm² flask (manufactured by Greiner) and allowed to stand atroom temperature for 1 hour to adhere the cells to the flask. A 1 mlportion of a solution containing about 1 to 3×10⁸ PFU/ml of recombinantvirus particles obtained in the above step (3) from the transfer vectorspVL1393/Flt 7N and pVL1393/Flt 3N was added to the flask to carry outinfection at room temperature for 2 hours. The culture supernatant wasremoved and 30 ml of fresh EX-CELL™ 400 medium was added to carry out 3to 4 days of culturing at 27° C. After completion of the culturing, theculture supernatant was collected and centrifuged at 1,500×g for 10minutes to obtain a supernatant fluid.

A column was packed with about 60 ml of heparin-Sepharose CL-6B gel(manufactured by Pharmacia Biotech AB) and washed with 600 ml of 20 mMTris-HCl (pH 7.5) buffer at a flow rate of 0.5 ml/minute. After thewashing, 1,000 ml of the culture medium containing soluble human VEGFreceptors Flt-1 7N and Flt-1 3N, which had been prepared in theabove-described manner, was passed through the heparin-Sepharose CL-6Bcolumn at a flow rate of 0.5 ml/minute. After washing with 600 ml of 20mM Tris-HCl (pH 7.5) buffer at a flow rate of 0.5 ml/minute, 600 ml of20 mM Tris-HCl (pH 7.5) buffer having a density gradient of 0 M to 1.1 MNaCl was passed through the column to carry out elution of the proteinsadsorbed to the heparin-Sepharose, and the eluate was fractionated in 8ml portions. Proteins contained in each fraction were analyzed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE), and 60 to 80 ml offractions containing soluble human VEGF receptors Flt-1 7N and Flt-1 3Nwere collected and concentrated using CentriPrep 10 (manufactured byAmicon) After the concentration, soluble human Flt-1 7N and Flt-1 3Nwere obtained as solutions of 5 ml and 13 ml, respectively (proteinconcentrations were 331 μg/ml and 204 μg/ml).

(5) Confirmation of the Purity of Soluble Human VEGF Receptors Flt-1 7Nand Flt-1 3N

Purity of the thus purified soluble human VEGF receptors Flt-1 7N andFlt-1 3N was confirmed by SDS-PAGE. The SDS-PAGE was carried out inaccordance with a known method [Anticancer Research, 12: 1121 (1992)].Using a 5 to 20% gradient gel (manufactured by Atto) as the gel,electrophoresis of Flt-1 7N and Flt-1 3N, each 2 μg as protein per lane,was carried out under reducing conditions, and the resulting gel wasstained with Coomassie Brilliant Blue. The results are shown in FIG. 3.Purity of Flt-1 7N and Flt-1 3N was found to be 95% or more.

(6) Purification of Control Antigen Protein of Soluble Human VEGFReceptors Flt-1 7N and Flt-1 3N

The control antigen protein (negative control protein) of soluble humanVEGF receptors Flt-1 7N and Flt-1 3N was obtained in the followingmanner. A 4×10⁷ portion of High Five cells were suspended in 30 ml ofEX-CELL™ 400 medium (manufactured by JRH Biosciences) contained in a 175cm² flask (manufactured by Greiner), allowed to stand at roomtemperature for 1 hour to adhere the cells to the flask and thencultured at 27° C. for 3 to 4 days. After completion of the culturing,the culture supernatant was collected and centrifuged at 1,500×g for 10minutes to obtain a supernatant fluid.

A column was packed with about 20 ml of heparin-Sepharose CL-6B gel(manufactured by Pharmacia Biotech AB) and washed with 200 ml of 20 mMTris-HCl (pH 7.5) buffer at a flow rate of 0.5 ml/minute. After thewashing, 500 ml of the culture medium of High Five cells was passedthrough the heparin-Sepharose CL-6B column at a flow rate of 0.5ml/minute. After washing with 200 ml of 20 mM Tris-HCl (pH 7.5) bufferat a flow rate of 0.5 ml/minute, 200 ml of 20 mM Tris-HCl (pH 7.5)buffer containing 1 M NaCl was passed through the column to carry outelution of the protein adsorbed to the heparin-Sepharose. The 1 M NaClelution fraction was concentrated using CentriPrep 10 (manufactured byAmicon) to obtain 7 ml of the control antigen protein (867 μg/ml asprotein concentration).

(7) Confirmation of Human VEGF Binding Activity of Soluble Human VEGFReceptors Flt-1 7N and Flt-1 3N

The human VEGF binding activity of soluble human VEGF receptors Flt-1 7Nand Flt-1 3N was confirmed in the following manner.

Methanol was dispensed in 100 μl portions into wells of a 96 wellImmobilon™-P Filtration Plate (manufactured by Millipore) to give ahydrophilic nature to the PVDF membrane on the bottom of the plate Afterwashing with water, the soluble human Flt-1 7N diluted to aconcentration of 2 μg/ml was dispensed in 50 μl/well portions andallowed to stand overnight at 4° C. for its adsorption. After washing,PBS containing 1% bovine serum albumin (BSA) was dispensed in 100μl/well portions and the reaction was carried out at room temperaturefor 1 hour to block the remained active groups. After washing with PBS,each of the purified soluble human VEGF receptors Flt-1 7N and Flt-1 3Nobtained in the above-described step (4) was dispensed in 50 μl/wellportions (final concentration, 1 to 1,000 ng/ml) and then ¹²⁵I-labeledhuman VEGF (final concentration, 3 ng/ml: produced by Amersham) wasdispensed in 50 μl/well portions, subsequently carrying out the reactionat room temperature for 1.5 hours. After washing with 0.05% Tween-PBS,the wells were dried at 50° C., and Microscinti-0 (manufactured byPackard) was dispensed in 20 μl/well portions to measure theradioactivity of the ¹²⁵I-labeled human VEGF linked to each well usingTop Count (manufactured by Packard).

The results are shown in FIG. 4. It was shown that soluble human VEGFreceptors Flt-1 7N and Flt-1 3N inhibit binding of ¹²⁵I-labeled humanVEGF to soluble human VEGF receptor Flt-1 7N in a concentrationdependent manner. Since the soluble human VEGF receptors Flt-1 7N andFlt-1 3N showed similar degree of the human VEGF binding activity, itwas revealed that the human VEGF binds to the Flt-1 3N moiety (the 1stto 338th positions from the N-terminal amino acid including signalsequence).

(8) Expression of Human VEGF in Insect Cells

The human VEGF was obtained in the following manner. A 4×10⁷ portion ofHigh Five cells were suspended in 30 ml of EX-CELL™ 400 medium(manufactured by JRH Biosciences) contained in a 175 cm² flask(manufactured by Greiner) and allowed to stand at room temperature for 1hour to adhere the cells to the flask. A 1 ml portion of a solutioncontaining about 1 to 3×10⁸ PFU/ml of human VEGF recombinant baculovirusparticles obtained in accordance with the known method [Cell Growth &Differentiation, 7: 213 (1996)] was added to the flask to carry outinfection at room temperature for 2 hours. The culture supernatant wasremoved and 30 ml of fresh EX-CELL™ 400 medium was added to carry out 3to 4 days of culturing at 27° C. After completion of the culturing, theculture supernatant was collected and centrifuged at 1,500×g for 10minutes to obtain a supernatant fluid.

A column was packed with about 40 ml of heparin-Sepharose CL-6B gel(manufactured by Pharmacia Biotech AB) and washed with 400 ml of 20 mMTris-HCl (pH 7.5) buffer at a flow rate of 0.5 ml/minute. After washing,1,500 ml of the culture medium containing human VEGF prepared in theabove-described manner was passed through the heparin-Sepharose CL-6Bcolumn at a flow rate of 0.5 ml/minute. After washing with 400 ml of 20mM Tris-HCl (pH 7.5) buffer at a flow rate of 0.5 ml/minute, 120 ml ofeach of 20 mM Tris-HCl (pH 7.5) buffers containing 0.2 M, 0.5 M and 1 MNaCl was passed through the column in that order to carry out stepwiseelution of the proteins adsorbed to the heparin-Sepharose, and theeluate was fractionated in 8 ml portions. Proteins contained in eachfraction were analyzed by SDS polyacrylamide gel electrophoresis, and120 ml of fractions (0.5 to 1 M NaCl fractions) containing human VEGFwere collected. After concentration using CentriPrep-10 (manufactured byAmicon), human VEGF was obtained as 4 ml of solution (proteinconcentration, 1.2 mg/ml).

2. Immunization of Animals and Preparation of Antibody Producing Cells

A 50 μg portion of each of the antigens obtained in the above-describedstep 1-(4) was administered, together with 2 mg of aluminum hydroxidegel and 1×10⁹ cells of pertussis vaccine (manufactured by Chiba Serum.Institute), into 5-week-old female BALB/c mice (SLC Japan), B6C3F1 mice(Charles River Japan) or female SD rats (SLC Japan), and, starting on 2weeks thereafter, 10 to 50 μg of the protein was administered once aweek for a total of four times. Also, 1×10⁷ of NIH3T3-Flt-1 cells wereadministered 6 times into three, 5 week old female BALB/c (SLC Japan)mice. Blood samples were collected from the fundus of the eye or thecaudal vein, their serum antibody titers were examined by the enzymeimmunoassay described in the following, and spleens were excised frommice or rats showing sufficient antibody titer 3 days after the finalimmunization. In this connection, immunization was not induced in the5-week-old female BALB/c to which NIH3T3-Flt-1 cells were administered,so that the antibody titer upon soluble Flt-1 7N was not increased.

The thus excised spleen was cut to pieces in MEM medium (manufactured byNissui Pharmaceutical), unbound using a pair of forceps and thencentrifuged (1,200 rpm for 5 minutes). The resulting supernatant wasdiscarded, and the thus obtained sediment was treated with Tris-ammoniumchloride buffer (pH 7.65) for 1 to 2 minutes to eliminate erythrocytes,washed three times with MEM medium and used in cell fusion.

3. Enzyme Immunoassay

With regard to the measurement of antisera derived from mice or ratsimmunized with the soluble human Flt-1 7N and Flt-1 3N obtained in theabove-described step 1-(4) and culture supernatants of hybridomas, thesoluble human VEGF receptors Flt-1 7N and Flt-1 3N obtained from theinsect cell culture supernatant of 1-(4) were used as antigens. A 1 to10 μg/ml PBS-diluted solution of each of the soluble human VEGFreceptors Flt-1 7N and Flt-1 3N and the heparin column adsorptionfraction of High Five cell culture supernatant obtained in theabove-described step 1-(6) as a control antigen was dispensed in 50μl/well portions into a 96 well plate for EIA (manufactured by Greiner)and allowed to stand overnight at 4° C. for coating. After washing, PBScontaining 1% bovine serum albumin (BSA) was dispensed in 100 μl/wellportions and the reaction was carried out at room temperature for 1 hourto block the remained active groups. After discarding 1% BSA-PBS,antiserum of immunized mouse or immunized rat and culture supernatant ofa hybridoma were dispensed in 50 μl/well portions to carry out thereaction for 2 hours. After washing with 0.05% Tween-PBS,peroxidase-labeled rabbit anti-mouse immunoglobulin orperoxidase-labeled rabbit anti-rat immunoglobulin (both manufactured byDAKO) was dispensed in 50 μl/well portions and the reaction was carriedout at room temperature for 1 hour, the plate was washed with 0.05%Tween-PBS and then color development was caused using ABTS substratesolution (2,2-azinobis(3-ethylbenzothiazole-6-sulfonic acid) ammoniumsalt) to measure maximum absorbance at OD415 nm using H max(manufactured by Molecular Devices).

4. Preparation of Mouse Myeloma Cells

8-Azaguanine-resistant mouse myeloma cell line P3U1 was cultured usingnormal medium to secure 2×10⁷ or more of the cells for use in cellfusion as the parent cell line.

5. Preparation of Hybridoma

The mouse spleen cells or rat spleen cells obtained in theabove-described section 2 and the myeloma cells obtained in the abovesection 4 were mixed to a ratio of 10:1 and centrifuged (1,200 rpm for 5minutes), the supernatant was discarded, the precipitated cells werethoroughly loosened to which, while stirring at 37° C., weresubsequently added a mixed solution of 2 g polyethylene glycol-1000(PEG-1000), 2 ml MEM medium and 0.7 ml DMSO in an amount of 0.2 to 1ml/10⁸ mouse myeloma cells and then 1 to 2 ml of MEM medium severaltimes at 1 to 2 minute intervals, and then the total volume was adjustedto 50 ml by adding MEM medium. After centrifugation (900 rpm for 5minutes), the supernatant was discarded and the thus obtained cells weregently loosened and then gently suspended in 100 ml of HAT medium byrepeated drawing up into and discharging from a graduated pipette.

The suspension was dispensed in 100 μl portions into wells of a 96 wellculture plate and cultured at 37° C. for 10 to 14 days in an atmosphereof 5% CO₂ in a 5% CO₂ incubator. The resulting culture supernatant wasexamined by the enzyme immunoassay method described in Example 1-3 toselect wells which reacted specifically with the soluble human VEGFreceptor Flt-1 7N or Flt-1 3N obtained in the above-described step 1-(4)but did not react with the control antigen obtained in the step 1-(6),and then cloning was repeated twice by changing the medium to HT mediumand normal medium to establish hybridomas capable of producinganti-human VEGF receptor Flt-1 monoclonal antibodies. The results areshown in the following table.

TABLE 1 The number of Screening Wells hybridomas Animal Head Immunogensource screened established Balb/c 3 NIH3T3-Flt-1 Flt 7N — — mouse SDrat 1 Flt 7N Flt 7N 1008 3 (KM1733, 1735, 1736) Balb/c 1 Flt 7N Flt 7N672 5 (KM1737, mouse 1739, 1740, 1742, 1743) SD rat 1 Flt 7N Flt 7N 11763 (KM1745, 1746, 1747) B3C3F1 1 Flt 7N Flt 3N 672 3 (KM1748, mouse 1749,1750) Balb/c 1 Flt 7N Flt 3N 420 3 (KM1730, mouse 1731, 1732)

When hybridomas obtained from one Balb/c mouse and two SD rats immunizedwith the soluble human VEGF receptor Flt-1 7N prepared in theabove-described step 1-(4) were screened for about 672 wells and about2,184 wells, respectively, using the soluble human VEGF receptor Flt-17N, respective 5 clones and 6 clones of anti-human VEGF receptor Flt-1monoclonal antibodies were obtained, and they were named KM1737, KM1739,KM1740, KM1742 and KM1743 and KM1733, KM1735, KM1736, KM1745, KM1746 andKM1747, respectively. None of these clones showed the action to inhibitbinding of human VEGF to Flt-1 as shown in the following section 8.Additionally, KM1735, KM1736, KM1742, KM1743 and KM1745 reacted withhuman VEGF receptor Flt-1 expression cells by the immunocyte stainingmethod described in the following section 10, but the reaction wasextremely weak in comparison with KM1730, KM1731 and KM1732.

On the other hand, when hybridomas obtained from one B3C3F1 mouse andone Balb/c mouse immunized with the soluble human VEGF receptor Flt-1 7Nprepared in the above-described step 1-(4) were screened for about 672wells and about 420 wells, respectively, using the soluble human VEGFreceptor Flt-1 3N, 3 clones for each of anti-human VEGF receptor Flt-1monoclonal antibodies were obtained, and they were named KM1748, KM1749and KM1750 and KM1730, KM1731 and KM1732, respectively. Of these clones,three clones KM1732, KM1748 and KM1750 showed the action to inhibitbinding of human VEGF to Flt-1 as shown in the following section 8.Additionally, three clones KM1730, KM1731 and KM1732 reacted markedlystrongly with human VEGF receptor Flt-1 expression cells by theimmunocyte staining method described in the following section 10.

The antibody class of these monoclonal antibodies was determined byenzyme immunoassay using Subclass Typing Kit (manufactured by Zymed).The results are shown in the following table.

TABLE 2 Monoclonal antibody Antibody subclass KM1733 mouse IgG2a KM1735rat IgG1 KM1736 rat IgG2a KM1737 mouse IgG1 KM1739 mouse IgG1 KM1740mouse IgG1 KM1742 mouse IgG1 KM1743 mouse IgG1 KM1745 rat IgG2a KM1746rat IgG1 KM1747 rat IgG1 KM1748 mouse IgG2b KM1749 mouse IgG1 KM1750mouse IgG2b KM1730 mouse IgG1 KM1731 mouse IgG2a KM1732 mouse IgG1

All of the monoclonal antibodies established in the present inventionwere IgG class.

6. Purification of Monoclonal Antibody

The hybridomas obtained in the above section 5 were respectivelyadministered to pristane-treated female nude mice (Balb/c) of 8 weeks ofage by intraperitoneal injection at a dose of 5 to 20×10⁶ cells peranimal. The hybridomas caused ascites tumor formation in 10 to 21 days.The ascitic fluid was collected from each ascitic fluid-carrying mouse(1 to 8 ml per animal), centrifuged (3,000 rpm for 5 minutes) forremoving solid matter and then purified by a caprylic acid precipitationmethod (Antibodies—A Laboratory Manual).

7. Confirmation of the Specificity of Monoclonal Antibodies

Specificity of the anti-human VEGF receptor Flt-1 monoclonal antibodiesdescribed in the above-described section 5 was confirmed using theenzyme immunoassay method described in the above-described section 3.

The results are shown in FIG. 5. Among the monoclonal antibodiesobtained by preparing hybridomas from mice and rats immunized with Flt-17N and selected using Flt-1 7N (KM1733, KM1735, KM1736, KM1737, KM1739,KM1740, KM1742, KM1743, KM1745, KM1746 and KM1747), only KM1740 reactedwith Flt-1 7N and Flt-1 3N, revealing that it recognizes an epitopewhich is present in a region of the 1 st to 338th positions from theN-terminal amino acid of Flt-1 (including signal sequence). Since theremaining 10 clones reacted with Flt-1 7N but not with Flt-1 3N, it wasrevealed that they recognize an epitope which is present in a region ofthe 339th to 750th positions from the N-terminal amino acid of Flt-1(including signal sequence) On the other hand, since all of themonoclonal antibodies obtained by preparing hybridomas from miceimmunized with Flt-1 7N and selecting using Flt-1 3N (KM1748, KM1749,KM1750, KM1730, KM1731 and KM1732) reacted with Flt-1 7N and Flt-1 3N,it was revealed that they recognize an epitope which is present in aregion of the 1st to 338th positions from the N-terminal amino acid ofFlt-1 (including signal sequence).

8. Confirmation of the Activity of Anti-Flt-1 Monoclonal Antibodies toInhibit Binding of a Human VEGF to a Human VEGF Receptor Flt-1

The activity of the anti-human VEGF receptor Flt-1 monoclonal antibodiesdescribed in the above-described section 5 to inhibit binding of humanVEGF to human VEGF receptor Flt-1 was confirmed in the following manner.

Methanol was dispensed in 100 μl portions into wells of a 96 wellMultiScreen-IP Plate (manufactured by Millipore) to give hydrophilicnature to the PVDF membrane on the bottom of the plate. After washingwith water, the soluble human VEGF receptor Flt-1 7N diluted with PBS toa concentration of 1.6 μg/ml was dispensed in 50 μl/well portions andthen allowed to stand overnight at 4° C. for its adsorption. Afterwashing, PBS containing 1% bovine serum albumin (BSA) is dispensed in 50μl/well portions and the reaction was carried out at room temperaturefor 1 hour to block the remained active groups. After washing with PBS,each hybridoma culture supernatant or a purified monoclonal antibodydiluted with 1% BSA-PBS containing 0.5 M NaCl (0.01 to 7.29 μg/ml) wasdispensed in 50 μl/well portions and then 3 ng/ml of ¹²⁵I-labeled humanVEGF (manufactured by Amersham) was dispensed in 50 μl/well portions,subsequently carrying out the reaction at room temperature for 1.5hours. After washing with 0.05% Tween-PBS, the wells were dried at 50°C., and Microscinti-0 (manufactured by Packard) was dispensed in 30μl/well portions to measure the radioactivity of the ¹²⁵I-labeled humanVEGF linked to each well using Top Count (manufactured by Packard).

Results of the examination of activities of hybridoma culturesupernatants are shown in FIG. 6. Among 17 established monoclonalantibodies, three monoclonal antibodies, KM1748, KM1750 and KM1732inhibited binding of human VEGF to human VEGF receptor Flt-1 atinhibition ratios of 62.6%, 66.3% and 83.1%, respectively.

In general, screening of monoclonal antibody producing hybridomas iscarried out using the same protein as the antigen used as the immunogen.A total of 11 monoclonal antibodies selected using Flt-1 7N as theimmunogen showed no binding inhibition activity, and, among 6 monoclonalantibodies selected using Flt-1 3N (KM1748, KM1749, KM1750, KM1730,KM1731 and KM1732), KM1748, KM1750 and KM1732 showed the bindinginhibition activity. It was an unexpected effect that monoclonalantibodies having the binding inhibition activity were obtained by theuse of Flt-1 3N in the screening of hybridomas. Thus, it was revealedthat Flt-1 3N is markedly important in establishing monoclonalantibodies having the binding inhibition activity.

FIG. 7 shows results of the examination of binding inhibition activityusing purified anti-Flt-1 monoclonal antibodies KM1732, KM1748 andKM1750. These antibodies KM1732, KM1748 and KM1750 inhibited binding ofhuman VEGF to human VEGF receptor Flt-1 in a concentration dependentmanner. Concentrations of KM1732, KM1748 and KM1750, which indicate 50%inhibition of the binding of human VEGF to human VEGF receptor Flt-1(IC₅₀), were 1.1, 1.3 and 2.0 μg/ml, respectively. On the other hand, ananti-sialyl-Le^(a) monoclonal antibody KM231 of a mouse IgG1 class[Anticancer Research, 10: 1579 (1990)] used as the control showed noinhibition activity.

9. Confirmation of the Activity of Anti-Flt-1 Monoclonal Antibodies toInhibit Binding of Human VEGF to Human VEGF Receptor Flt-1 ExpressionCells

The activity of the anti-human VEGF receptor Flt-1 monoclonal antibodiesKM1732, KM1748 and KM1750 to inhibit binding of human VEGF to human VEGFreceptor Flt-1 was confirmed in the following manner.

PBS containing 1% bovine serum albumin (BSA) was dispensed in 100 μlportions into wells of a 96 well MultiScreen-HV Plate (manufactured byMillipore), the reaction was carried out at room temperature for 1 hourto block the active groups in the wells and then NIH3T3-Flt-1 cellssuspended in 1% BSA-PBS containing 0.05% NaN₃ were dispensed in 5×10⁴cells/well portions. After washing with 1% BSA-PBS, a purifiedmonoclonal antibody (0.01 to 7.29 μg/ml) was dispensed in 50 μl/wellportions and then 3 ng/ml of ¹²⁵I-labeled human VEGF (manufactured byAmersham) was dispensed in 50 μl/well portions and the reaction wascarried out under cooling for 2 hours. After washing with PBS, the wellswere dried at 50° C., and Microscinti-0 (manufactured by Packard) wasdispensed in 30 μl/well portions to measure the radioactivity of the¹²⁵I-labeled human VEGF linked to each well using Top Count(manufactured by Packard).

FIG. 8 shows results of the examination of binding inhibition activityusing purified anti-Flt-1 monoclonal antibodies KM1732, KM1748 andKM1750. These antibodies KM1732, KM1748 and KM1750 inhibited binding ofhuman VEGF to NIH3T3-Flt-1 cells in a concentration dependent manner.Concentrations of KM1732, KM1748 and KM1750, which indicate 50%inhibition of the binding of human VEGF to NIH3T3-Flt-1 cells (IC₅₀),were 0.050, 0.037 and 0.041 μg/ml, respectively. On the other hand, theanti-sialyl-Le^(a) monoclonal antibody KM231 of a mouse IgG1 class usedas the control showed no inhibition activity.

10. Confirmation of the Reactivity of Monoclonal Antibodies with HumanVEGF Receptor Flt-1 Expression Cells

Specificity of the anti-human VEGF receptor Flt-1 monoclonal antibodiesdescribed in the above-described section 5 was confirmed usingimmunocyte staining method in accordance the following procedure.

A total of 5×10⁵ cells of each of human VEGF receptor Flt-1 expressionNIH3T3 cells (NIH3T3-Flt-1) and control NIH3T3 cells (NIH3T3-Neo)[Oncogene, 10: 135 (1995)] were suspended in 100 μl of a buffer solutionfor immunocyte staining use (PBS containing 1% BSA, 0.02% EDTA and 0.05%sodium azide) and dispensed in a round bottom 96 well plate. Aftercentrifugation at 4° C. and at 350×g for 1 minute, the supernatant fluidwas discarded and the resulting cells were mixed with 50 μl of ahybridoma culture supernatant or purified antibody (10 μg/ml) andreaction was carried out at 4° C. for 30 minutes. After the reaction,200 μl of the buffer solution for immunocyte staining use was added toeach well, and the cells were washed by centrifugation at 4° C. and350×g for 1 minute followed by discarding the resulting supernatant.After repeating this washing step twice, the cells were mixed with 50 μlof the buffer solution for immunocyte staining use containing 1 μg/ml ofan FITC-labeled anti-mouse immunoglobulin antibody or FITC-labeledanti-rat immunoglobulin antibody (manufactured by Wako Pure ChemicalIndustries), and the reaction was carried out at 4° C. for 30 minutes.After this reaction, the above-described washing step was repeated threetimes and then analysis was carried out using Flow Cytometer(manufactured by Coulter).

The results are shown in FIG. 9. The anti-human VEGF receptor Flt-1monoclonal antibodies KM1730, KM1731 and KM1732 did not react with thecontrol cells but specifically reacted in significant amounts with theFlt-1 expression cells. Neither, the anti-human VEGF receptor Flt-1monoclonal antibody KM1748 (10 μg/ml) nor the hybridoma culturesupernatant KM1748 reacted with the control cells. Each specificallyreacted in significant amounts with the Flt-1 expression cells (B). Asthe results, it was discovered that the monoclonal antibodies KM1730,KM1731, KM1732, KM1748 and KM1750 specifically recognize the human VEGFreceptor Flt-1 on the cell surface. On the other hand, KM1735, KM1736,KM1742, KM1743 and KM1745 only weakly reacted with the human VEGFreceptor Flt-1 expression cells in comparison with KM1730, KM1731,KM1732, KM1748 and KM1750.

11. Detection of Human VEGF Receptor Flt-1 by Western Blotting UsingMonoclonal Antibody

Cell membrane components were prepared from NIH3T3-Flt-1 cells andcontrol NIH3T3 cells (NIH3T3-Neo) in accordance with a known method[Cancer Research, 46: 4438 (1986)] and electrophoresed by the SDS-PAGEmethod. The SDS-PAGE was carried out in accordance with a known method[Anticancer Research, 12: 1121 (1992)] by subjecting 15 μg, as proteinper lane, of the cell membrane components to the electrophoresis using a5 to 20% gradient gel (manufactured by Atto) under reducing conditions.The thus treated proteins were transferred to a PVDF membrane inaccordance with a known method [Anticancer Research, 12: 1121 (1992)].Next, the PVDF membrane was allowed to react with PBS containing 1% BSAat room temperature for 30 minutes to carry out blocking and then toreact with the culture supernatant of the anti-human VEGF receptor Flt-1monoclonal antibody KM1737 overnight at 4° C. The thus treated membranewas washed with PBS containing 0.05% Tween and then allowed to reactwith peroxidase-labeled goat anti-mouse IgG (5,000 times dilution:produced by Chemicon) at room temperature for 2 hours. After washingwith 0.05% Tween-containing PBS, bands to which the anti-human VEGFreceptor Flt-1 monoclonal antibody KM1737 was linked were detected usingECL™ Western blotting detection reagents (manufactured by Amersham).

The results are shown in FIG. 10. It was confirmed that the anti-humanVEGF receptor Flt-1 monoclonal antibody KM1737 can detect the human VEGFreceptor Flt-1 of 180 kilo dalton in molecular weight expressed in theNIH3T3-Flt-1 cells.

12. Detection of Soluble Human VEGF Receptor Flt-1 Using MonoclonalAntibody

The anti-human VEGF receptor Flt-1 monoclonal antibody KM1732 wasdiluted with PBS to a concentration of 10 μg/ml and dispensed in 50μl/well portions into a 96 well plate for EIA (manufactured by Greiner)and allowed to stand overnight at 4° C. for coating. After washing, PBScontaining 1% bovine serum albumin (BSA) was dispensed in 100 μl/wellportions and the reaction was carried out at room temperature for 1 hourto block the remained active groups. After discarding 1% BSA-PBS, thepurified soluble human VEGF receptors Flt-1 7N and Flt-1 3N obtained inthe above-described step 1-(4) and diluted with 1% BSA-PBS to aconcentration of 1,000 to 0.0056 ng/ml were allowed to react with theantibody overnight at 4° C. After washing with 0.05% Tween-PBS, theanti-human VEGF receptor Flt-1 monoclonal antibody KM1730 labeled withbiotin by a known method [Enzyme Antibody Method: published by GakusaiKikaku (1985)] was diluted with 1% BSA-PBS to a concentration of 0.1μg/ml and dispensed in 50 μl/well portions to carry out the reaction atroom temperature for 2 hours. After washing with 0.05% Tween-PBS,avidin-labeled peroxidase (manufactured by Vector) diluted 4,000 timeswith 1% BSA-PBS was dispensed in 50 μl/well portions to carry out thereaction at room temperature for 1 hour. After washing with 0.05%Tween-PBS, color development was caused using ABTS substrate solution[2,2-azinobis(3-ethylbenzothiazole-6-sulfonic acid) ammonium salt] tomeasure absorbance at OD415 nm using H max (manufactured by MolecularDevices).

The results are shown in FIG. 11. As the results, it was found that thesoluble human VEGF receptors Flt-1 3N and Flt-1 7N can be measured fromminimum concentrations of 0.46 ng/ml and 1.37 ng/ml, respectively, bythe use of the anti-human VEGF receptor Flt-1 monoclonal antibody KM1732and the biotin-labeled anti-human VEGF receptor Flt-1 monoclonalantibody KM1730.

13. Confirmation of the Reactivity of Monoclonal Antibodies with HumanVascular Endothelial Cells HUVEC

The reactivity of anti-human VEGF receptor Flt-1 monoclonal antibodiesdescribed in the above-described section 5 with human vascularendothelial cells HUVEC was confirmed by immunocyte staining in thefollowing manner.

A total of 2×10⁵ cells of human umbilical vein endothelial cells (HUVEC)were suspended in 100 μl of a buffer solution for immunocyte staininguse (PBS containing 1% BSA, 0.02% EDTA and 0.05% sodium azide) anddispensed in a round bottom 96 well plate. After centrifugation at 4° C.and at 350×g for 1 minute, the supernatant fluid was discarded and theresulting cells were mixed with 50 μl (10 μg/ml) of each of biotinatedpurified antibodies KM1730 and KM1750 and control antibodies thereof,individually, and subsequently incubated at 4° C. for 30 minutes. As thecontrol antibody of KM1730, an anti-MxA monoclonal antibody KM1135 (WO96/05230) of IgG1 type which is the same subclass of KM1730 was used. Asthe control antibody of KM1750, an anti-T cell receptor γ chainmonoclonal antibody KM365 (Japanese Published Unexamined PatentApplication No. 491/90) of IgG2b which is the same subclass as KM1750was used. Thereafter, 200 μl of the buffer solution for immunocytestaining use was added to each well, and the cells were washed bycarrying out centrifugation at 4° C. and at 350×g for 1 minute and thenthe resulting supernatant was discarded. After again repeating thiswashing step twice, the cells were mixed with 20 μl of the buffersolution for immunocyte staining use containing 5 μg/ml in concentrationof Avidin-PE (Streptoavidin-R-Phycoerythrin) (manufactured by Gibco),and the reaction was carried out at 4° C. for 30 minutes. After thereaction, the above-described washing step was repeated three times andthen the analysis was carried out using Flow Cytometer (manufactured byCoulter).

The results are shown in FIG. 12. The anti-human VEGF receptor Flt-1monoclonal antibodies KM1730 and KM1750 reacted with HUVEC when comparedwith their control antibodies. These results demonstrate that themonoclonal antibodies KM1730 and KM1750 can detect human VEGF receptorFlt-1 on human vascular endothelial cells.

14. Increase of the Expression Quantity of Flt-1 on HUVEC by VEGFStimulation

As a model of vascular endothelial cells in an angiogenesis region,changes in the expression of human VEGF receptor Flt-1 before and afterstimulation with VEGF were examined using the anti-human VEGF receptorFlt-1 monoclonal antibody KM1730 in accordance with the followingprocedure.

A total of 4 to 6×10⁵ cells of each of four lots of HUVEC (lot #4031,#4102, #2477 and #4723; purchased from Clonetics) were suspended in 20ml of a medium (manufactured by KURABO) (control medium) comprising E-BMmedium further supplemented with 5% fetal bovine serum (FBS), 10 ng/mlof human recombinant type epidermal growth factor (hEGF), 1 μg/ml ofhydrocortisone, 50 μg/ml of gentamicin and 50 ng/ml of amphotericin, andthe suspension was further mixed with 1.2 μg/ml of bovine brain extract(BBE) (manufactured by KURABO) as a growth factor and cultured at 37° C.for 2 to 3 days. When the cells were proliferated into 1 to 2×10⁶ cells,the medium was removed and replaced with 20 ml of fresh control mediumto carry out a total of 2 days of culturing. After the culturing for 1day, human VEGF was added to a final concentration of 5 ng/ml, and thecells after the additional culturing for 1 day were used asVEGF-stimulated cells. Cells cultured for 2 days without adding VEGFwere used as control cells (VEGF-non-stimulated cells) After theculturing, the cells were collected to examine reactivity of theanti-human VEGF receptor Flt-1 monoclonal antibody KM1730 by theimmunocyte staining method in accordance with the procedure described inthe above section 13.

Results of the examination of its reactivity with the lot #2477 HUVECare shown in FIG. 13. KM1730 reacted with VEGF-non-stimulated HUVEC butmore strongly with VEGF-stimulated HUVEC. Reactivity of the controlantibody KM1135 did not change independent of the VEGF stimulation ornon-stimulation. FIG. 14 shows changes in the Flt-1 expression in fourlots of HUVEC (lot #4031, #4102, #2477 and #4723) by VEGF stimulationThe expression quantity of Flt-1 which can express the reactivity ofKM1730 as an index is shown as a relative value when reactivity of thecontrol antibody is defined as 1. It was revealed that all of the fourlots of HUVEC can express Flt-1 by VEGF non-stimulation, and theexpression quantity of Flt-1 increases by the VEGF stimulation.

The increase of the expression quantity of Flt-1 and the reactivity ofanti-Flt-1 monoclonal antibody in the VEGF-stimulated human vascularendothelial cells HUVEC as a model of angiogenesis shows that themonoclonal antibody is useful for the diagnosis or treatment of diseasesin which their morbid states progress by the acceleration ofangiogenesis caused by VEGF, such as tumors, rheumatoid arthritis,diabetic retinopathy, and the like.

EXAMPLE 2

1. Isolation and Analysis of cDNA Encoding Anti-human VEGF ReceptorFlt-1 Mouse Monoclonal Antibody

(1) Preparation of mRNA From Hybridoma Capable of Producing Anti-humanVEGF Receptor Flt-1 Mouse Monoclonal Antibody

Using a mRNA extraction kit, Fast Track manufactured by Invitrogen, andin accordance with the manual attached to the kit, mRNA was preparedfrom 1×10⁸ cells of each of hybridomas capable of producing anti-humanVEGF receptor Flt-1 mouse monoclonal antibodies KM1732 and KM1750obtained in Example 1 (FERM BP-5698 and FERM BP-5-700, respectively).

(2) Preparation of Heavy Chain and Light Chain cDNA Libraries ofHybridoma Capable of Producing Anti-human VEGF Receptor Flt-1 MouseMonoclonal Antibody

Using cDNA Synthesis Kit (manufactured by Pharmacia Biotech) and inaccordance with the manual attached to the kit, cDNA having EcoRI-NotIadapter on both termini was synthesized from 5 μg of each of KM1732 andKM1750 mRNA samples obtained in 1(1) of Example 2. About 6 μg of each ofthe thus prepared cDNA samples was dissolved in 10 μl of sterilizedwater and fractionated by agarose gel electrophoresis to recover about0.1 μg of a cDNA fragment of about 1.5 kb which corresponds to the heavychain (hereinafter referred to as “H chain”) of the IgG type antibodyand similar amount of a cDNA fragment of about 1.0 kb that correspondsto the light chain (hereinafter referred to as “L chain”) thereof. Next,0.1 μg of the cDNA fragment of about 1.5 kb, 0.1 μg of the cDNA fragmentof about 1.0 kb and 1 μg of Lambda ZAPII Vector (a preparation obtainedby digesting Lambda ZAPII Vector with EcoRI and then treating it withcalf intestine alkaline phosphatase: manufactured by Stratagene) weredissolved in 11.5 μl of a T4 ligase buffer solution (manufactured byTakara Shuzo Co., Ltd.), and the thus prepared solution was mixed with175 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) andincubated at 12° C. for 24 hours and then at room temperature for 2hours. Thereafter, a 4 μl portion of each of the reaction solutions waspackaged in lambda phage using Gigapack Gold Packaging Kit (manufacturedby Stratagene) in accordance with the conventional method [MolecularCloning, 2.95, Cold Spring Harbor Laboratory (1989)], and Escherichiacoli XL1-Blue [Biotechniques, 5: 376 (1987)] attached to Gigapack GoldPackaging Kit (manufactured by Stratagene) was infected with theresulting phage in accordance with the conventional method [MolecularCloning, 2.95-107, Cold Spring Harbor Laboratory (1989)] to obtain about4×10³ phage clones as each of the H chain cDNA libraries and L chaincDNA libraries of KM1732 and KM17540.

(3) Cloning of cDNA Encoding H Chain and L Chain of Hybridoma Capable ofProducing Anti-human VEGF Receptor Flt-1 Mouse Monoclonal Antibody

Respective phage particles prepared in the step 1-(2) of Example 2 werefixed on nitrocellulose filters in accordance with the conventionalmethod [Molecular Cloning, 2.12, Cold Spring Harbor Laboratory (1989)].Each of the resulting nitrocellulose filters was treated in accordancewith the manual attached to ECL direct nucleic acid labelling anddetection systems (manufactured by Amersham) to obtain phage cloneswhich strongly bonded to a probe cDNA encoding the constant region(hereinafter referred to as “C region”) of mouse immunoglobulin {cDNAencoding H chain is a fragment of mouse Cγ1 cDNA [Cell, 18: 559 (1979)]and cDNA encoding L chain is a fragment of mouse Cκ cDNA [Cell, 22: 197(1980)]}. Thereafter, the phage clones were transformed into plasmidpBluescript SK(−) in accordance with the manual attached to Lambda ZAPIIVector (manufactured by Stratagene), thus finally obtaining arecombinant plasmid KM1732HA2 containing cDNA encoding the H chain ofKM1732, a recombinant plasmid KM1732L2-1 containing cDNA encoding Lchain of KM1732, a recombinant plasmid KM1750H2-1 containing cDNAencoding the H chain of KM1750 and a recombinant plasmid KM1750L3-1containing cDNA encoding L chain of KM1750. Escherichia coli XL1-BlueMRF′/KM1732HA2 containing the recombinant plasmid KM1732HA2, Escherichiacoli XL1-Blue MRF′/KM1732L2-1 containing the recombinant plasmidKM1732L2-1, Escherichia coli XL1-Blue MRF′/KM1750H2-1 containing therecombinant plasmid KM1750H2-1 and Escherichia coli XL1-BlueMRF′/KM1750L3-1 containing the recombinant plasmid KM1750L3-1 have beendeposited on May 14, 1998, in National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, and have beenassigned the designations as FERM BP-6354, FERM BP-6352, FERM BP-6353and FERM BP-6355, respectively.

(4) Determination of Variable Region Nucleotide Sequence of cDNAEncoding H Chain and L Chain of Anti-human VEGF Receptor Flt-1 MouseMonoclonal Antibody

Variable region (hereinafter referred to as “V region”) nucleotidesequence of each of the cDNA samples obtained in the step 1-(3) ofExample 2 encoding H chain and L chain of anti-human VEGF receptor Flt-1mouse monoclonal antibody was determined by reacting 0.1 μg of each ofthe obtained plasmid samples in accordance with the manual attached toBigDye Terminator Cycle Sequencing Ready Reaction Kit (manufactured byApplied Biosystems) and then carrying out electrophoresis using ABIPRISM™ 377 (manufactured by Applied Biosystems) Based on the thusdetermined nucleotide sequence of each cDNA, amino acid sequences of theV region of H chain (hereinafter referred to as “VH”) and the V regionof L chain (hereinafter referred to as “VL”) of KM1732 and KM1750 weredetermined. The nucleotide sequence and amino acid sequence of the VH ofKM1732 are shown in SEQ ID NOS:5 and 86, respectively, those of the VLof KM1732 are shown in SEQ ID NOS:6 and 87, respectively, those of theVH of KM1750 are shown in SEQ ID NOS:7 and 88, respectively, and thoseof the VL of KM1750 in SEQ ID NOS:8 and 89, respectively.

(5) Identification of CDR Sequences of H Chain and L Chain of Anti-humanVEGF Receptor Flt-1 Mouse Monoclonal Antibody

Based on the amino acid sequences of the VH and VL of anti-human VEGFreceptor Flt-1 mouse monoclonal antibody determined in the step 1-(4) ofExample 2, CDR sequences of respective VH and VL were identified bycomparing them with known antibody V region amino acid sequences(Sequences of Proteins of Immunological Interest). The amino acidsequences of CDR 1, 2 and 3 of the VH of KM1732 are shown in SEQ IDNOS:9, 10 and 11, respectively; those of CDR 1, 2 and 3 of the VL ofKM1732 are shown in SEQ ID NOS:12, 13 and 14, respectively; those of CDR1, 2 and 3 of the VH of KM1750 are shown in SEQ ID NOS:15, 16 and 17,respectively; and those of CDR 1, 2 and 3 of the VL of KM1750 are shownin SEQ ID NOS:18, 19 and 20, respectively.

2. Production of Anti-human VEGF Receptor Flt-1 Human ChimericAntibodies

Anti-human VEGF receptor Flt-1 human chimeric antibodies KM2532 andKM2550 originated from the anti-human VEGF receptor Flt-1 mousemonoclonal antibodies KM1732 and KM1750 having the activity to inhibitbiological activities of human VEGF receptor Flt-1 were produced in thefollowing manner.

(1) Construction of Expression Vector pKANTEX1732 of Anti-human VEGFReceptor Flt-1 Human Chimeric Antibody

The expression vector pKANTEX1732 of anti-human VEGF receptor Flt-1human chimeric antibody was constructed in the following manner using atandem cassette vector pKANTEX93 for humanized antibody expression usedescribed in WO 97/10354 and the plasmids KM1732HA2 and KM1732L2-1obtained in Example 2-1.

A 3 μg portion of plasmid pBluescript SK(−) (manufactured by Stratagene)was added to 10 μl of a buffer solution comprising 10 mM Tris-HCl (pH7.5), 10 mM magnesium chloride and 1 mM DTT, 10 units of a restrictionenzyme ApaI (manufactured by Takara Shuzo Co., Ltd.) were further addedthereto, and then the reaction was carried out at 37° C. for 1 hour. Thereaction solution was precipitated with ethanol, the thus obtainedprecipitate was added to 10 μl of a buffer solution comprising 50 mMTris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1mM DTT, 100 μg/ml bovine serum albumin (hereinafter referred to as“BSA”) and 0.01% Triton X-100, 10 units of a restriction enzyme NotI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was fractionated by agarose gel electrophoresis to recoverabout 2 μg of an ApaI-NotI fragment of about 2.95 kb. Next, 5 μg of theplasmid KM1732HA2 was added to 10 μl of a buffer solution comprising 20mM Tris-ECl (pH 7.9), 10 mM magnesium acetate, 50 mM potassium acetate,1 mM DTT and 100 μg/ml BSA, 10 units of a restriction enzyme NlaIV(manufactured by New England Biolabs) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was precipitated with ethanol, the thus obtained precipitatewas added to 10 μl of a buffer solution comprising 50 mM Tris-ECl (pH7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100μg/ml BSA and 0.01% Triton X-100, 10 units of a restriction enzyme NotI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was fractionated by agarose gel electrophoresis to recoverabout 0.5 μg of an NlaIV-NotI fragment of about 0.41 kb. Next, asynthetic DNA having the nucleotide sequence of SEQ ID NO:21 or 22 wassynthesized (manufactured by Sawady Technology), and a 0.3 μg portion ofeach synthetic DNA was added to 15 μl of sterilized water and heated at65° C. for 5 minutes. The reaction solution was allowed to stand at roomtemperature for 30 minutes, mixed with 2 μl of a 10-fold buffer solution[500 mM Tris-HCl (pH 7.6), 100 mm magnesium chloride and 50 mM DTT] and2 μl of 10 mM ATP and then with 10 units of T4 polynucleotide kinase(manufactured by Takara Shuzo Co., Ltd.), and the mixture was allowed toreact at 37° C. for 30 minutes to phosphorylating the 5′ end.

A 0.1 μg portion of the ApaI-NotI fragment derived from plasmidpBluescript SK(−), 0.1 μg of the NlaIV-NotI fragment derived fromplasmid KM1732HA2 and 0.05 μg of the phosphorylated synthetic DNA,obtained by the just described procedures, were added to 10 μl in totalvolume of sterilized water and ligated using DNA Ligation Kit Ver. 2(manufactured by Takara Shuzo Co., Ltd.) in accordance with the manualattached thereto. Escherichia coli DH5α (manufactured by TOYOBO CO.,LTD.) was transformed with the thus prepared recombinant plasmid DNAsolution to obtain a plasmid pBS1732H shown in FIG. 15.

Next, 3 μg portion of plasmid phKM1259LV0 described in WO 97/10354 wasadded to 10 μl of a buffer solution comprising 50 mM Tris-HCl (pH 7.5),100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/mlBSA, 10 units of a restriction enzyme EcoRI (manufactured by TakaraShuzo Co., Ltd.) and 10 units of a restriction enzyme SplI (manufacturedby Takara Shuzo Co., Ltd.) were further added thereto, and then thereaction was carried out at 37° C. for 1 hour. The reaction solution wasfractionated by agarose gel electrophoresis to recover about 2 μg of anEcoRI-SplI fragment of about 2.95 kb. Next, 5 μg of the plasmidKM1732L2-1 was added to 10 μl of a buffer solution comprising 10 mMTris-HCl (pH 7.5), 100 mM magnesium chloride and 1 mM DTT, 10 units of arestriction enzyme MboII (manufactured by TOYOBO CO., LTD.) were furtheradded thereto, and then the reaction was carried out at 37° C. for 1hour. The reaction solution was precipitated with ethanol, the thusobtained precipitate was added to 10 μl of a buffer solution comprising50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesiumchloride and 1 mM DTT, 10 units of a restriction enzyme EcoRI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was fractionated by agarose gel electrophoresis to recoverabout 0.5 μg of a MboII-EcoRI fragment of about 0.38 kb. Next, asynthetic DNA having the nucleotide sequence of SEQ ID NO:23 or 24 wassynthesized (manufactured by Sawady Technology), and a 0.3 μg portion ofeach synthetic DNA was added to 15 μl of sterilized water and heated at65° C. for 5 minutes. The reaction solution was allowed to stand at roomtemperature for 30 minutes, mixed with 2 μl of a 10-fold buffer solution[500 mM Tris-HCl (pH 7.6), 100 mM magnesium chloride and 50 mM DTT] and2 μl of 10 mM ATP and then with 10 units of T4 polynucleotide kinase(manufactured by Takara Shuzo Co., Ltd.), and the mixture was allowed toreact at 37° C. for 30 minutes to phosphorylate the 5′ end.

A 0.1 μg portion of the EcoRI-SplI fragment derived from plasmidphKM1259LV0, 0.1 μg of the MboII-EcoRI fragment derived from plasmidKM1732L2-1 and 0.05 μg of the phosphorylated synthetic DNA, obtained bythe just described procedures, were added to 10 μl in total volume ofsterilized water and ligated using DNA Ligation Kit Ver. 2 (manufacturedby Takara Shuzo Co., Ltd.) in accordance with the manual attachedthereto. Escherichia coli DH5α (manufactured by TOYOBO CO., LTD.) wastransformed with the thus prepared recombinant plasmid DNA solution toobtain a plasmid pBS1732L shown in FIG. 16.

Next, 3 μg of the vector pKANTEX93 for humanized antibody expression wasadded to 10 μl of a buffer solution comprising 10 mM Tris-HCl (pH 7.5),10 mM magnesium chloride and 1 mM DTT, 10 units of a restriction enzymeApaI (manufactured by Takara Shuzo Co., Ltd.) were further addedthereto, and then the reaction was carried out at 37° C. for 1 hour. Thereaction solution was precipitated with ethanol, the thus obtainedprecipitate was added to 10 μl of a buffer solution comprising 50 mMTris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1mM DTT, 100 μg/ml BSA and 0.01% Triton X-100, 10 units of a restrictionenzyme NotI (manufactured by Takara Shuzo Co., Ltd.) were further addedthereto, and then the reaction was carried out at 37° C. for 1 hour. Thereaction solution was fractionated by agarose gel electrophoresis torecover about 1 μg of an ApaI-NotI fragment of about 12.75 kb. Next, 5μg of the plasmid pBS1732H obtained in the foregoing was added to 10 μlof a buffer solution comprising 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, 10 units of a restriction enzyme ApaI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was precipitated with ethanol, the thus obtained precipitatewas added to 10 μl of a buffer solution comprising 50 mM Tris-HCl (pH7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100μg/ml BSA and 0.01% Triton X-100, 10 units of a restriction enzyme NotI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was fractionated by agarose gel electrophoresis to recoverabout 0.5 μg of an ApaI-NotI fragment of about 0.46 kb.

A 0.1 μg portion of the ApaI-NotI fragment derived from the vectorpKANTEX93 for humanized antibody expression and 0.1 μg of the ApaI-NotIfragment derived from the plasmid pBS1732H, obtained by the justdescribed procedures, were added to 10 μl in total volume of sterilizedwater and ligated using DNA Ligation Kit Ver. 2 (manufactured by TakaraShuzo Co., Ltd.) in accordance with the manual attached thereto.Escherichia coli DH5α (manufactured by TOYOBO CO., LTD.) was transformedwith the thus prepared recombinant plasmid DNA solution to obtain aplasmid pKANTEX1732H. shown in FIG. 17.

Next, 3 μg of the just obtained plasmid pKANTEX1732H was added to 10 μlof a buffer solution comprising 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 unitsof a restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.)and 10 units of a restriction enzyme SplI (manufactured by Takara ShuzoCo., Ltd.) which were further added thereto, and then the reaction wascarried out at 37° C. for 1 hour. The reaction solution was fractionatedby agarose gel electrophoresis to recover about 1 μg of an EcoRI-SplIfragment of about 13.20 kb. Next, 5 μg of the plasmid pBS1732L obtainedin the foregoing was added to 10 μl of a buffer solution comprising 50mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride,1 mM DTT and 100 μg/ml BSA, 10 units of a restriction enzyme EcoRI(manufactured by Takara Shuzo Co., Ltd.) and 10 units of a restrictionenzyme SplI (manufactured by Takara Shuzo Co., Ltd.) were further addedthereto, and then the reaction was carried out at 37° C. for 1 hour. Thereaction solution was fractionated by agarose gel electrophoresis torecover about 0.5 μg of an EcoRI-SplI fragment of about 0.39 kb.

A 0.1 μg portion of the EcoRI-SplI fragment derived from the plasmidpKANTEX1732H and 0.1 μg of the EcoRI-SplI fragment derived from theplasmid pBS1732L, obtained by the just described procedures, were addedto 10 μl in total volume of sterilized water and ligated using DNALigation Kit Ver. 2 (manufactured by Takara Shuzo Co., Ltd.) inaccordance with the manual attached thereto. Escherichia coli DH5α(manufactured by TOYOBO CO., LTD.) was transformed with the thusprepared recombinant plasmid DNA solution to obtain a plasmidpKANTEX1732 shown in FIG. 18.

(2) Construction of Expression Vector pKANTEX1750 of Anti-human VEGFReceptor Flt-1 Human Chimeric Antibody

The expression vector pKANTEX1750 of anti-human VEGF receptor Flt-1human chimeric antibody was constructed in the following manner using atandem cassette vector pKANTEX93 for humanized antibody expressiondescribed in WO 97/10354 and the plasmids KM1750H2-1 and KM1750L3-1obtained in Example 2-1.

A 5 μg portion of the plasmid KM1750H2-1 was added to 10 μl of a buffersolution comprising 33 mM Tris-HCl (pH 7.9), 10 mM magnesium acetate, 66mM potassium acetate and 100 μg/ml BSA, 10 units of a restriction enzymeAlw26I (manufactured by New England Biolabs) were further added thereto,and then the reaction was carried out at 37° C. for 1 hour. The reactionsolution was precipitated with ethanol, the thus obtained precipitatewas added to 10 μl of a buffer solution comprising 50 mM Tris-HCl (pH7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100μg/ml BSA and 0.01% Triton X-100, 10 units of a restriction enzyme NotI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was fractionated by agarose gel electrophoresis to recoverabout 0.5 μg of an Alw261I-NotI fragment of about 0.41 kb. Next, asynthetic DNA having the nucleotide sequence of SEQ ID NO:25 or 26 wassynthesized (manufactured by Sawady Technology), and a 0.3 μg portion ofeach synthetic DNA was added to 15 μl of sterilized water and heated at65° C. for 5 minutes. The reaction solution was allowed to stand at roomtemperature for 30 minutes, mixed with 2 μl of a 10-fold buffer solution[500 mM Tris-HCl (pH 7.6), 100 mM magnesium chloride and 50 mM DTT] and2 μl of 10 mM ATP and then with 10 units of T4 polynucleotide kinase(manufactured by Takara Shuzo Co., Ltd.), and the mixture was allowed toreact at 37° C. for 30 minutes to phosphorylate the 5′ end.

A 0.1 μg portion of the ApaI-NotI fragment derived from plasmidpBluescript SK(−) in the step 2-(1) of Example 2, 0.1 μg of theAlwI-NotI fragment derived from plasmid KM1750H2-1 and 0.05 μg of thephosphorylated synthetic DNA were added to 10 μl in total volume ofsterilized water and ligated using DNA Ligation Kit Ver. 2 (manufacturedby Takara Shuzo Co., Ltd.) in accordance with the manual attachedthereto. Escherichia coli DH5α (manufactured by TOYOBO CO., LTD.) wastransformed with the thus prepared recombinant plasmid DNA solution toobtain a plasmid pBS1750H shown in FIG. 19.

Next, 5 μg of the plasmid KM1750L3-1 was added to 10 μl of a buffersolution comprising 100 mM Tris-HCl (pH 8.8), 440 mM sodium chloride, 12mM magnesium chloride, 14 mM 2-mercaptoethanol and 200 μg/ml BSA, 10units of a restriction enzyme MaeII (manufactured byBoehringer-Mannheim) were further added thereto, and then the reactionwas carried out at 50° C. for 1 hour. The reaction solution wasprecipitated with ethanol, the thus obtained precipitate was added to 10μl of a buffer solution comprising 50 mM Tris-HCl (pH 7.5), 100 mMsodium chloride, 10 mM magnesium chloride and 1 mM DTT, 10 units of arestriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.) werefurther added thereto, and then the reaction was carried out at 37° C.for 1 hour. The reaction solution was fractionated by agarose gelelectrophoresis to recover about 0.5 μg of a MaeII-EcoRI fragment ofabout 0.38 kb. Next, a synthetic DNA having the nucleotide sequence ofSEQ ID NO:27 or 28 was synthesized (manufactured by Sawady Technology),and a 0.3 μg portion of each synthetic DNA was added to 15 μl ofsterilized water and heated at 65° C. for 5 minutes. The reactionsolution was allowed to stand at room temperature for 30 minutes, mixedwith 2 μl of a 10-fold buffer solution [500 mM Tris-HCl (pH 7.6), 100 mMmagnesium chloride and 50 mM DTT] and 2 μl of 10 mM ATP and then with 10units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co.,Ltd.), and the mixture was allowed to react at 37° C. for 30 minutes tophosphrylate the 5′ end.

A 0.1 μg portion of the EcoRI-SplI fragment derived from plasmidpphKM1259LV0 in the step 2-(1) of Example 2, 0.1 μg of the MaeII-EcoRIfragment derived from plasmid KM1750L3-1 and 0.05 μg of thephosphorylated synthetic DNA were added to 10 μl in total volume ofsterilized water and ligated using DNA Ligation Kit Ver. 2 (manufacturedby Takara Shuzo Co., Ltd.) in accordance with the manual attachedthereto. Escherichia coli DH5α (manufactured by TOYOBO CO., LTD.) wastransformed with the thus prepared recombinant plasmid DNA solution toobtain a plasmid pBS1750L shown in FIG. 20.

Next, 5 μg of the plasmid pBS1750H obtained in the foregoing was addedto 10 μl of a buffer solution comprising 10 mM Tris-HCl (pH 7.5), 10 mMmagnesium chloride and 1 mM DTT, 10 units of a restriction enzyme ApaI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was precipitated with ethanol, the thus obtained precipitatewas added to 10 μl of a buffer solution comprising 50 mM Tris-HCl (pH7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100μg/ml BSA and 0.01% Triton X-100, 10 units of a restriction enzyme NotI(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionsolution was fractionated by agarose gel electrophoresis to recoverabout 0.5 μg of an ApaI-NotI fragment of about 0.46 kb.

A 0.1 μg portion of the ApaI-NotI fragment derived in the step 2-(1) ofExample 2 from a tandem cassette vector pKANTEX93 for humanized antibodyexpression and 0.1 μg of the ApaI-NotI fragment derived from plasmidpBS1750H were added to 10 μl in total volume of sterilized water andligated using DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo Co.,Ltd.) in accordance with the manual attached thereto. Escherichia coliDH5α (manufactured by TOYOBO CO., LTD.) was transformed with the thusprepared recombinant plasmid DNA solution to obtain a plasmidpKANTEX1750H shown in FIG. 21.

Next, 3 μg of the just obtained plasmid pKANTEX1750H was added to 10 μlof a buffer solution comprising 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 unitsof a restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.)and 10 units of a restriction enzyme SplI (manufactured by Takara ShuzoCo., Ltd.) were further added thereto, and then the reaction was carriedout at 37° C. for 1 hour. The reaction solution was fractionated byagarose gel electrophoresis to recover about 1 μg of an EcoRI-SplIfragment of about 13.20 kb. Next, 5 μg of the plasmid pBS1750L obtainedin the foregoing was added to 10 μl of a buffer solution comprising 50mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride,1 mM DTT and 100 μg/ml BSA, 10 units of a restriction enzyme EcoRI(manufactured by Takara Shuzo Co., Ltd.) and 10 units of a restrictionenzyme SplI (manufactured by Takara Shuzo Co., Ltd.) were further addedthereto, and then the reaction was carried out at 37° C. for 1 hour. Thereaction solution was fractionated by agarose gel electrophoresis torecover about 0.5 μg of an EcoRI-SplI fragment of about 0.39 kb.

A 0.1 μg portion of the EcoRI-SplI fragment derived from plasmidpKANTEX1750H and 0.1 μg of the EcoRI-SplI fragment derived from plasmidpBS1750L, obtained by the just described procedures, were added to 10 μlin total volume of sterilized water and ligated using DNA Ligation KitVer. 2 (manufactured by Takara Shuzo Co., Ltd.) in accordance with themanual attached thereto. Escherichia coli DH5α (manufactured by TOYOBOCO., LTD.) was transformed with the thus prepared recombinant plasmidDNA solution to obtain a plasmid pKANTEX1750 shown in FIG. 22.

(3) Expression of Anti-human VEGF Receptor Flt-1 Human ChimericAntibodies in Rat Myeloma YB2/0 Cells (ATCC CRL1581) Using pKANTEX1732and pKANTEX1750

Introduction of anti-human VEGF receptor Flt-1 human chimeric antibodyexpression vectors pKANTEX1732 and pKANTEX1750 into YB2/0 cells wascarried out by electroporation in accordance with the method of Miyagiet al. [Cytotechnology, 3: 133 (1990)].

A 5 μg portion of each of pKANTEX1732 and pKANTEX1750 obtained in thesteps 2-(1) and (2) of Example 2 was introduced into 4×10⁶ YB2/0 cellswhich were subsequently suspended in 40 ml of RPMI 1640-FCS(10) medium[RPMI 1640 medium (manufactured by Nissui Pharmaceutical) containing 10%fetal calf serum (FCS)] and dispensed in 200 μl portions into wells of a96 well microtiter plate (manufactured by Sumilon). After 24 hours ofculturing at 37° C. in a 5% CO₂ incubator, geneticin (hereinafterreferred to as “G 418”, manufactured by Gibco) was added to a finalconcentration of 0.5 mg/ml, and the culturing was continued for 1 to 2weeks. When colonies of G 418-resistant transformants were formed andbecame confluent, the culture supernatant was recovered from each well,and the activity of anti-human VEGF receptor Flt-1 human chimericantibody in the supernatant was measured by the following enzymeimmunoassay.

Enzyme Immunoassay

Soluble human VEGF receptor Flt-1 7N was prepared in accordance with themethod of Tanaka et al. [Japanese Journal of Cancer Research, 88:867-876 (1997)]. The soluble human VEGF receptor Flt-1 7N diluted to 1μg/ml with PBS was dispensed in 50 μl portions into wells of a 96 wellplate for EIA (manufactured by Greiner) and allowed to stand overnightat 4° C. for its adsorption. After washing with PBS, 100 μl of PBScontaining 1% BSA (hereinafter referred to as “1% BSA-PBS”) was added toeach well, and the remaining active groups was blocked by carrying outthe reaction at room temperature for 1 hour. After discarding 1%BSA-PBS, the culture supernatant of each transformant was dispensed in50 μl portions into the resulting wells to carry out the reaction atroom temperature for 1 hour. After washing the plate with PBS containing0.05% Tween 20 (hereinafter referred to as “0.05% Tween-PBS”), aperoxidase-labeled anti-human IgG antibody (manufactured by AmericanQuarex) which had been diluted 3,000 times with 1% BSA-PBS was dispensedin 50 μl portions into the wells to carry out the reaction at roomtemperature for 1 hour, the resulting plate was washed with 0.5%Tween-PBS, and then an ABTS [ammonium2,2-azinobis(3-ethylbenzothiazole-6-sulfonate)] substrate solution wasdispensed in 50 μl portions into the wells to develop color and measureits absorbance at OD_(415 nm) using Emax (manufactured by MolecularDevices).

In order to increase productivity, each of the transformants whichshowed the anti-human VEGF receptor Flt-1 human chimeric antibodyactivity in the culture supernatant was suspended in RPMI 1640-FCS(10)medium supplemented with 0.5 mg/ml of G 418 and 50 nM of methotrexate(hereinafter referred to as “MTX”, manufactured by Sigma) and culturedat 37° C. for 1 to 2 weeks in a 5% CO₂ incubator to induce transformantshaving 50 nM MTX resistance. When the transformants became confluent inthe wells, the anti-human VEGF receptor Flt-1 human chimeric antibodyactivity in the culture supernatants was measured by the aforementionedenzyme immunoassay. Transformants in which the activity was found werecultured similarly by further increasing the MTX concentration to 100 nMand then to 200 nM to obtain transformants which can grow in RPMI1640-FCS(10) medium containing 0.5 mg/ml of G 418 and 200 nM of MTX andare able to produce large amounts of anti-human VEGF receptor Flt-1human chimeric antibodies. The thus obtained transformants weresubjected to two times of cloning by limiting dilution to obtain finaltransformant cell strains capable of producing anti-human VEGF receptorFlt-1 human chimeric antibodies. KM2532 can be cited as an example ofthe transformant cell strain obtained by introducing the expressionvector pKANTEX1732, namely a transformant which produces an anti-humanVEGF receptor Flt-1 human chimeric antibody originated from theanti-human VEGF receptor Flt-1 mouse monoclonal antibody KM1732, and theanti-human VEGF receptor Flt-1 human chimeric antibody produced by thistransformant was named KM2532. Also, KM2550 can be cited as an exampleof the transformant cell strain obtained by introducing the expressionvector pKANTEX1750, namely a transformant which produces an anti-humanVEGF receptor Flt-1 human chimeric antibody originated from theanti-human VEGF receptor Flt-1 mouse monoclonal antibody KM1750, and theanti-human VEGF receptor Flt-1 human chimeric antibody produced by thistransformant was named KM2550. The productivity of anti-human VEGFreceptor Flt-1 human chimeric antibodies by the thus clonedtransformants was found to be about 5 μg/10⁶ cells/24 hours.

(4) Purification of Anti-human VEGF Receptor Flt-1 Human ChimericAntibodies From Culture Supernatants

Each of the anti-human VEGF receptor Flt-1 human chimeric antibodyproducing strains KM2532 and KM2550 obtained in 2-(3) of Example 2 wassuspended in GIT medium (manufactured by Japan Pharmaceutical)supplemented with 0.5 mg/ml of G 418 and 200 nM of MTX, to a density of1 to 2×10⁵ cells/ml, and dispensed in 200 ml portions into a total offive 175 cm² flasks (manufactured by Greiner). When the cells becameconfluent after 5 to 7 days of culturing at 37° C. in a 5% CO₂incubator, about 1.0 liter of each culture supernatant was recovered. Acolumn was packed with about 1 ml of ProSep A (manufactured byBioprocessing) and washed with 10 ml of 1 M glycine-0.15 M NaCl (pH 8.6)at a flow rate of 1 ml/minute. After the washing, 1,700 ml or 1,800 mlof the culture supernatant prepared in the above containing theanti-human VEGF receptor Flt-1 human chimeric antibody KM2532 or KM2550was passed through the ProSep A column at a flow rate of 70 ml/hour.This was further washed with 10 ml of 1 M glycine-0.15 M NaCl (pH 8.6)at a flow rate of 1 ml/minute, again washed stepwise with 4 ml of 50 mMcitrate buffer of pH 6, 5 and 4, and then 7 ml of 50 mM citrate buffer(pH 3.0) was passed through the column to elute the human chimericantibody. As the results, 0.4 mg and 0.3 mg of the anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 wereobtained.

The thus purified anti-human VEGF receptor Flt-1 human chimericantibodies KM2532 and KM2550 were analyzed by SDS-PAGE in accordancewith a known method [Anticancer Research, 12: 1121 (1992)]. A the gel, 5to 20% gradient gel (manufactured by Atto Corp.) was used, a lowmolecular weight protein molecular marker “Daiichi” II and a highmolecular weight protein molecular marker “Daiichi” III (bothmanufactured by Daiichi Pure Chemicals) were used as molecular markers,and 2.5 μg as protein per lane of each of the anti-human VEGF receptorFlt-1 human chimeric antibodies KM2532 and KM2550 was electrophoresedunder reducing and non-reducing conditions and stained with CoomassieBrilliant Blue. The results are shown in FIG. 23. The human chimericantibodies KM2532 and KM2550 showed a band of IgG at a positioncorresponding to a molecular weight of about 150 kDa under thenon-reducing condition, and a band of H chain at about 50 kDa and a bandof L chain at about 25 kDa were found under the reducing condition. Thisresult coincided with that the IgG type antibody is separated into two Hchains and L chains under the reducing conditions caused by the cuttingof intermolecular disulfide bond and present as a molecule of 150 kDa.

(5) Binding Activity of Anti-human VEGF Receptor Flt-1 Human ChimericAntibodies Upon Human VEGF Receptor Flt-1

Binding activity of the purified anti-human VEGF receptor Flt-1 humanchimeric antibodies KM2532 and KM2550 upon human VEGF receptor Flt-1 wasconfirmed by the following procedure.

Soluble human VEGF receptor Flt-1 7N was prepared in accordance with themethod of Tanaka et al. [Japanese Journal of Cancer Research, 88:867-876 (1997)].

First, the binding activity was examined using an immunoassay method byfixing amount of the soluble human VEGF receptor Flt-1 7N to be adsorbedon a 96 well plate for EIA and varying concentration of the humanchimeric antibody to be added. The soluble human VEGF receptor Flt-1 7Ndiluted to 1 μg/ml with PBS was dispensed in 50 μl portions into wellsof a 96 well plate for EIA (manufactured by Greiner) and allowed tostand overnight at 4° C. for its adsorption. After washing the platewith PBS, 100 μl of 1% BSA-PBS was added to each well, and the remainingactive groups was blocked by carrying out the reaction at roomtemperature for 1 hour. After discarding 1% BSA-PBS, each of thepurified anti-human VEGF receptor Flt-1 human chimeric antibodies KM2532and KM2550 having a concentration of from 0.152 to 333 ng/ml wasdispensed in 50 μl portions into the resulting wells to carry out thereaction at room temperature for 1 hour. After washing the plate with0.05% Tween-PBS, a peroxidase-labeled anti-human IgG antibody(manufactured by American Corlex) which had been diluted 3,000 timeswith 1% BSA-PBS was dispensed in 50 μl portions into the wells to carryout the reaction at room temperature for 1 hour, the resulting plate waswashed with 0.5% Tween-PBS, and then an ABTS [ammonium2,2-azinobis(3-ethylbenzothiazole-6-sulfonate)] substrate solution wasdispensed in 50 μl portions into the wells to develop color and measureits absorbance at OD_(415 nm) using Emax (manufactured by MolecularDevices).

The results are shown in FIG. 24 (A). The anti-human VEGF receptor Flt-1human chimeric antibodies KM2532 and KM2550 bound to the human VEGFreceptor Flt-1 7N in an antibody concentration dependent fashion. Inaddition, the anti-human VEGF receptor Flt-1 human chimeric antibodiesKM2532 and KM2550 showed almost the same binding activity.

Next, the binding activity of human chimeric antibodies was examinedusing an immunoassay method by varying amount of the soluble human Flt-17N to be adsorbed on a 96 well plate for EIA. The soluble human VEGFreceptor Flt-1 7N diluted to a concentration of from 0.04 to 10 μg/mlwith PBS was dispensed in 50 μl portions into wells of a 96 well platefor EIA (manufactured by Greiner) and allowed to stand overnight at 4°C. for its adsorption. After washing the plate with PBS, 100 μl of 1%BSA-PBS was added to each well, and the remaining active groups wasblocked-by carrying out the reaction at room temperature for 1 hour.After discarding 1% BSA-PBS, each of the purified anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 having aconcentration of 10 μg/ml was dispensed in 50 μl portions into theresulting wells to carry out the reaction at room temperature for 1hour. After washing the plate with 0.05% Tween-PBS, a peroxidase-labeledanti-human IgG antibody (manufactured by American Corlex) which had beendiluted 3,000 times with 1% BSA-PBS was dispensed in 50 μl portions intothe wells to carry out the reaction at room temperature for 1 hour, theresulting plate was washed with 0.5% Tween-PBS, and then an ABTSammonium 2,2-azinobis(3-ethylbenzothiazole-6-sulfonate) substratesolution was dispensed in 50 μl portions into the wells to develop colorand measure its absorbance at OD_(415 nm) using Emax (manufactured byMolecular Devices).

The results are shown in FIG. 24 (B). Each of the anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 showed itsbinding activity dependently upon the concentration of the soluble humanVEGF receptor Flt-1 7N adsorbed on the plate. In addition, theanti-human VEGF receptor Flt-1 human chimeric antibodies KM2532 andKM2550 showed almost the same binding activity.

Next, the activity of anti-human VEGF receptor Flt-1 human chimericantibodies to inhibit binding of human VEGF with human VEGF receptorFlt-1 was examined in the following manner. Methanol was dispensed in100 μl portions into wells of a 96 well multi-screen IP plate(manufactured by Millipore) to change PVDF membrane on the plate bottominto hydrophilic nature. After washing the plate with water, the solublehuman VEGF receptor Flt-1 7N which had been diluted to 0.2 μg/ml withPBS was dispensed in 50 μl portions into the wells and allowed to standovernight at 4° C. for its adsorption. After washing the plate with PBS,50 μl of 1% BSA-PBS was added to each well, and the remaining activegroups was blocked by carrying out the reaction at room temperature for1 hour. After washing the plate with PBS, each of the purifiedanti-human VEGF receptor Flt-1 human chimeric antibodies KM2532 andKM2550 and purified anti-human VEGF receptor Flt-1 mouse monoclonalantibodies KM1732 and KM1750, which had been diluted with 1% BSA-PBS toa concentration of from 0.004 to 2 μg/ml, was dispensed in 50 μlportions into the resulting wells and then 3 ng/ml of ¹²⁵I-labeled humanVEGF (manufactured by Amersham) was dispensed in 50 μl portions into thewells to carry out the reaction at room temperature for 1.5 hours. Afterwashing the plate with 0.05% Tween-PBS, the wells were dried at 50° C.,Microscinti-0 (manufactured by Packard) was dispensed in 30 μl portionsinto the wells and then radioactivity of the ¹²⁵I-labeled human VEGFbonded on each well was measured using Top Count (manufactured byPackard). The results are shown in FIG. 25. As shown in FIG. 25, each ofthe anti-human VEGF receptor Flt-1 human chimeric antibodies KM2532 andKM2550 inhibited binding of the human VEGF with the human VEGF receptorFlt-1 in an antibody concentration dependent fashion. In addition, theactivity of the anti-human VEGF receptor Flt-1 human chimeric antibodiesKM2532 and KM2550 to inhibit binding of the human VEGF with the humanVEGF receptor Flt-1 was almost the same as that of the anti-human VEGFreceptor Flt-1 mouse monoclonal antibodies KM1732 and KM1750, thusshowing that the human chimeric antibodies maintain the activity ofmouse monoclonal antibodies.

(6) Analysis of Epitope which is Recognized by Anti-human VEGF ReceptorFlt-1 Human Chimeric Antibody

(6-1) Preparation of Soluble Human VEGF Receptor KDR 7N and SolubleHuman VEGF Receptor Chimeric Protein Flt-1 7N.K2

Soluble human VEGF receptor KDR 7N and soluble human VEGF receptorchimeric protein Flt-1 7N.K2 were produced in the following manner.

(6-2) Construction of Soluble Human VEGF Receptor KDR 7N ExpressionVector

A vector was prepared in the following manner for the expression of 19amino acids which constitute the signal peptide of human VEGF receptorKDR, a soluble human VEGF receptor KDR fragment (hereinafter referred toas “soluble human VEGF receptor KDR 7N”) which corresponds to 738 aminoacids counting from the N-terminal amino acid of its mature body and twoamino acid residues derived from a linker.

The soluble human VEGF receptor KDR 7N consists of the one to sevenimmunoglobulin-like moiety counting from the N-terminal side of theextracellular region of soluble human VEGF receptor KDR.

A cDNA clone which encodes complete length cDNA of human VEGF receptorKDR, BCMGS-neo-KDR [A. Sawano et al., Cell Growth & Differentiation, 7:213-221 (1996)], was digested with EcoRI, and a fragment of about 2.8 kbencoding the extracellular region and membrane-binding region of KDR wasintegrated into the EcoRI site of pUC18 to prepare pUC-KDR. The thusprepared pUC-KDR was digested with XhoI and treated with Klenow and thenan XbaI linker (SEQ ID NO:57) was inserted to prepare pUC-KDR-Xb. AnXbaI-BamHI fragment (2.3 kbp) of pUC-KDR-Xb was inserted into theXbaI-BamHI site of pBluescriptII KS(+) to prepare an SphI-BamHI fragment(5.2 kbp) into which were subsequently inserted synthetic linkers (SEQID NOS:58 and 59) containing SnaBI site to prepare pBS-KDR-Xb-S.

The thus obtained pBS-KDR-Xb-S was digested with SnaBI/BamHI, andsynthetic linkers (SEQ ID NOS:60 and 61) containing terminal codon andNotI site were inserted to prepare pBS-KDR-Xb-S-N. An XbaI-NotI fragment(2.3 kb) of pBS-KDR-Xb-S-N was inserted into downstream 5′ side XbaI and3′ side NotI site of the transcription initiation point of polyhedringene of baculovirus recombinant plasmid pVL1393 to obtain soluble humanVEGF receptor KDR 7N expression vector pVL-KDR-7N.

(6-3) Construction of Soluble Human VEGF Receptor Chimeric Protein Flt-17N.K2 Expression Vector

A vector was constructed in the following manner for the expression of achimeric protein Flt-1.K2 in which an amino acid sequence of from the100 position amino acid to the 204 position amino acid which correspondsto the second immunoglobulin-like moiety counting from the N-terminalside of the soluble human VEGF receptor Flt-1 7N that corresponds to 750amino acids counting from the N-terminal containing the signal sequenceof human VEGF receptor Flt-1 was replaced with an amino acid sequence offrom the 95 position amino acid to the 199 position amino acid whichcorresponds to the second immunoglobulin-like moiety of the solublehuman VEGF receptor KDR, via linkers of two amino acids (Gly-Ala andGly-Thr).

An EcoRI/HindIII fragment (1.9 kb) of human Flt-1 cDNA [Shibuya et al.,Oncogene, 5: 519 (1990)] was inserted into the EcoRI/HindIII site of avector M13mp19 to prepare pM13-flt. Escherichia coli XL1Blue wasinfected with the thus obtained pM13-flt and cultured, and ssDNA wasprepared from the resulting culture supernatant in accordance with amanual supplied by Takara Shuzo. Using an oligonucleotide of 56 bases(SEQ ID NO:62) and a site-specific mutation introducing kit Mutan K(manufactured by Takara Shuzo Co., Ltd.), site-specific mutation wascarried out using the thus obtained ssDNA as the template to prepare aplasmid pM13-flt′-D2N containing a Flt-1 mutant gene in which the regionencoding the second immunoglobulin-like moiety of the extracellularregion of Flt-1 was deleted. Next, using 10 ng of BCMGS-neo-KDR DNA [A.Sawano et al., Cell Growth & Differentiation, 7: 213 (1996)] as thetemplate, PCR was carried out by adding 2.5 units of TaqDNA polymeraseto 100 ml of a solution containing 10 pmol of each of primers havingnucleotide sequences shown in SEQ ID NOS:63 and 64, TaqDNA polymerasebuffer and 10 mM deoxynucleotide triphosphate. A serial reaction of 90seconds at 95° C., 90 seconds at 50° C. and 90 seconds at 72° C. wasrepeated 30 times, and the thus amplified DNA fragment encoding thesecond immunoglobulin-like moiety counting from the N-terminal side ofthe extracellular region of KDR was recovered. This fragment wasdigested with NarI/KpnI to obtain an NarI/KpnI fragment of 340 bp. Thethus obtained fragment and an SmaI/NarI fragment (0.5 kb) ofpM13-flt′-D2N were inserted into the SmaI/KpnI site of pBluescriptII toprepare pBS-flt-1′-KDR2N. Thereafter, a BamH/KpnI fragment (0.8 kb) ofpBS-flt-1′-KDR2N, a KpnI/MunI fragment (80 b) of pM13-flt′-D2N and anMunI/NotI fragment (1.5 kb) of pVL-flt-1 were inserted into theBamHII/NotI site of pVL1393 to obtain a plasmid PVL-fkf which carriesthe Flt-1 7N.K2 gene.

(6-4) Preparation of Recombinant Virus for the Expression of SolubleHuman VEGF Receptor KDR 7N and Soluble Human VEGF Receptor ChimericProtein Flt-1 7N.K2 in Insect Cells

In order to produce a protein in insect cells, it is necessary toprepare a recombinant virus into which the gene of interest isintegrated, and such a preparation process comprises a step in which acDNA called transfer vector which encodes the protein of interest isinserted into a special plasmid and a subsequent step in which a wildtype virus and the transfer vector are co-transfected into insect cellsto obtain the recombinant virus by homologous recombination. These stepswere carried out in the following manner using BaculoGold Starter Kitmanufactured by Fermingen (Product No. PM-21001K) in accordance with themanual attached thereto.

Recombinant baculovirus was prepared in the following manner by alipofectin method [Protein, Nucleic Acid and Enzyme, 37: 2701 (1992)],by introducing filamentous baculovirus DNA (BaculoGold baculovirus DNA,manufactured by Fermingen) and transfer vector DNA into cells of aninsect cell line Sf9 (manufactured by Fermingen) which had been culturedusing TMN-FH insect medium (manufactured by Fermingen).

A 1 μg portion of the expression vector prepared in the step (6-2) and20 ng of the filamentous baculovirus DNA were dissolved in 12 μl ofdistilled water, and a mixture consisting of 6 μl of lipofectin and 6 μlof distilled water was added to the resulting solution and allowed tostand at room temperature for 15 minutes. Separately from this, 1×10⁶ ofSf9 cells were suspended in 2 ml of Sf900-II medium (manufactured byGibco) and put into a plastic Petri dish for cell culture having adiameter of 35 mm. Entire portion of the aforementioned mixture solutionof plasmid DNA, filamentous baculovirus DNA and lipofectin was added tothe dish, the cells were cultured at 27° C. for 3 days and then a 1 mlportion of the culture supernatant containing recombinant virus wascollected. A 1 ml portion of fresh Sf900-II medium was added to theresulting Petri dish, and the cells were further cultured at 27° C. for3 days to obtain 1.5 ml of the culture supernatant containingrecombinant virus. Thereafter, the same procedure was repeated using theexpression vector obtained in the step (6-3).

Next, in order to use it in protein expression, each of the thusobtained recombinant viruses was grown in the following manner.

A total of 2×10⁷ Sf9 cells were suspended in 10 ml of Sf900-II medium,put into a 175 cm² flask (manufactured by Greiner) and then allowed tostand at room temperature for 1 hour to adhere the cells to the flask.Thereafter, the supernatant was discarded and 15 ml of fresh TMN-FHinsect medium and a 1 ml of portion of the aforementioned culturesupernatant containing recombinant virus were added to the flask tocarry out 3 days of culturing at 27° C. After the culturing, thesupernatant was centrifuged at 1,500×g for 10 minutes to remove cells toobtain a recombinant virus solution to be used in protein expression.

The titer of virus in the thus obtained recombinant virus solution wascalculated in accordance with the method described in BaculoGold StarterKit Manual (manufactured by Farmigen).

A total of 6×10⁶ Sf9 cells were suspended in 4 ml of Sf900-II medium,put into a plastic Petri dish for cell culture having a diameter of 60mm and then allowed to stand at room temperature for 1 hour to adherethe cells to the dish. Next, the supernatant was discarded, 400 μl offresh Sf900-II medium and the aforementioned recombinant virus solutiondiluted 1,000 times with Sf900-II medium in advance were added to thedish and allowed to stand at room temperature for 1 hour, and then themedium was discarded and 5 ml of a medium containing 1% low meltingpoint agarose (Agarplaque Agarose, manufactured by Farmigen) (a mediumprepared by mixing 1 ml of sterilized 5% Agarplaque Agarose aqueoussolution with 4 ml of TMN-FH insect medium and kept at 42° C.) waspoured into the Petri dish. After 15 minutes of standing at roomtemperature, the Petri dish was sealed with a vinyl tape to preventdrying and put into a sealing type plastic container to carry out 6 daysof culturing at 27° C. A 1 ml portion of PBS containing 0.01% NeutralRed was added to the Petri dish, and the culturing was continued for 1day to count the number of formed plaques. It was found by the aboveprocedure that each of the recombinant virus solutions contained about1×10⁷ plaque forming units (hereinafter referred to as “PFU”)/ml ofviral particles.

(6-5) Expression of Soluble Human VEGF Receptor KDR 7N and Soluble HumanVEGF Receptor Chimeric Protein Flt-1 7N.K2 in Insect Cells and TheirPurification

The soluble human VEGF receptor KDR 7N and soluble human VEGF receptorchimeric protein Flt-1 7N.K2 shown in (6-1) to (6-4) were obtained inthe following manner. A total of 4×10⁷ High Five cells were suspended in30 ml of EX-CELL™ 400 medium (manufactured by JRH Bioscience) containedin a 175 cm² flask (manufactured by Greiner) and adhered to the flask byallowing them to stand at room temperature for 1 hour. A 1 ml portion ofa solution containing about 1 to 3×10⁸ PFU/ml of the transfervector-originated recombinant virus obtained in (6-3) and (6-4) wasadded to the flask to carry out infection at room temperature for 2hours. The culture supernatant was discarded and replaced with 30 ml offresh EX-CELL 400 medium to carry out 3 to 4 days of culturing at 27° C.After completion of the culturing, the culture supernatant was collectedand centrifuged at 1,500×g for 10 minutes to recover the supernatant.

The soluble human VEGF receptor KDR 7N was purified in the followingmanner.

A column packed with 50 ml of DEAE-Sepharose CL-6B (manufactured byPharmacia Biotech) and a column packed with 40 ml of Heparin SepharoseCL-6B (manufactured by Pharmacia Biotech) were connected in series, theformer column on the inlet side and the latter on the outlet side, andwashed with 300 ml of 20 mM sodium phosphate buffer (pH 8). After thewashing, 400 to 800 ml of a culture solution containing soluble humanVEGF receptor KDR was passed through the columns at a flow rate of from50 to 100 ml/hour. The columns were again washed with 300 ml of 20 mMsodium phosphate buffer (pH 8) and then the adsorbed protein was elutedfrom the Heparin Sepharose CL-6B column by continuous density gradientusing 400 ml of 0 to 1 M NaCl/20 mM sodium phosphate buffer. The eluatewas fractionated in 7 ml portions, and 60 to 80 ml of fractionscontaining soluble human VEGF receptor KDR 7N were recovered byanalyzing the protein contained in each fraction by SDS-PAGE. The thusrecovered purification fractions were concentrated using CentriPrep 10(manufactured by Amicon) to obtain 4.8 ml of a soluble human VEGFreceptor KDR 7N solution (protein concentration and purity were 815μg/ml and 70 to 80%, respectively).

The soluble human VEGF receptor chimeric protein Flt-1 7N.K2 waspurified in the following manner.

A column was packed with about 20 ml of Heparin-Sepharose CL-6B gel(manufactured by Pharmacia Biotech AB) and washed with 100 ml of 20 mMTris-HCl buffer (pH 7.5) at a flow rate of 0.5 ml/minute. After thewashing, 900 ml of the culture solution containing soluble human VEGFreceptor chimeric protein Flt-1 7N.K2 prepared in the foregoing waspassed through the Heparin-Sepharose CL-6B column at a flow rate of 0.5ml/minute. The column was washed again with 100 ml of 20 mM Tris-HClbuffer (pH 7.5) at a flow rate of 0.5 ml/minute, and then 200 ml of 20mM Tris-HCl buffer (pH 7.5) containing NaCl having a density gradient offrom 0 to 1.5 M was passed through the column to elute the proteinadsorbed to Heparin-Sepharose, while the eluate was fractionated in 8 mlportions. By analyzing the protein contained in each fraction by SDSpolyacrylamide gel electrophoresis (SDS-PAGE), 50 to 70 ml of fractionscontaining soluble human VEGF receptor chimeric protein Flt-1 7N.K2 wererecovered and concentrated using CentriPrep 10 (manufactured by Amicon).After the concentration, human VEGF receptor chimeric protein Flt-17N.K2 was obtained as a 5.8 ml of solution (protein concentration, 588μg/ml; purity, approximately 70%).

(6-6) Analysis of Epitope which is Recognized by Anti-human VEGFReceptor Flt-1 Human Chimeric Antibody

The epitope which is recognized by purified anti-human VEGF receptorFlt-1 human chimeric antibodies KM2532 and KM2550 was analyzed in thefollowing manner.

Soluble human VEGF receptor Flt-1 7N (corresponds to a sequence from theN-terminal amino acid to the 750 position amino acid, including signalsequence), soluble human VEGF receptor Flt-1 3N (corresponds to asequence from the N-terminal amino acid to the 338 position amino acid,including signal sequence) and soluble human VEGF receptor Flt-1 2N(corresponds to a sequence from the N-terminal amino acid to the 223position amino acid, including signal sequence) were prepared inaccordance with the method of Tanaka et al. [Japanese Journal of CancerResearch, 88: 867-876 (1997)]. Soluble human VEGF receptor KDR 7N(corresponds to a sequence from the N-terminal amino acid to the 738position amino acid, does not include signal sequence) and soluble humanVEGF receptor chimeric protein Flt-1 7N.K2 (100 to 204 position aminoacids of human Flt-1 7N are replaced with 95 to 199 position amino acidsof human KDR via links of two amino acids) were prepared in accordancewith the method described in (6-5). Schematic illustration of each ofthe soluble human VEGF receptor derivatives used in the experiment isshown in FIG. 35.

First, the reactivity of anti-human VEGF receptor Flt-1 mouse monoclonalantibodies KM1732 and KM1750 and anti-human VEGF receptor Flt-1 humanchimeric antibodies KM2532 and KM2550 was compared using an immunoassaymethod. Each of Flt-1 7N, Flt-1 3N, Flt-1 2N, Flt-1 7N.K2 and KDR 7Ndiluted to 4 μg/ml with PBS was dispensed in 50 μl portions into wellsof a 96 well plate for EIA (manufactured by Greiner) and allowed tostand overnight at 4° C. for its adsorption. After washing the platewith PBS, 100 μl of 1% BSA-PBS was added to each well, and the remainingactive groups was blocked by carrying out the reaction at roomtemperature for 1 hour. After discarding 1% BSA-PBS, each of thepurified anti-human VEGF receptor Flt-1 mouse monoclonal antibodiesKM1732 and KM1750 or purified anti-human VEGF receptor Flt-1 humanchimeric antibodies KM2532 and KM2550, having a concentration of from0.1 μg/ml, was dispensed in 50 μl portions into the resulting wells tocarry out the reaction at room temperature for 1 hour. After washing theplate with 0.05% Tween-PBS, a peroxidase-labeled anti-mouse IgG antibody(manufactured by Dako) which had been diluted 400 times with 1% BSA-PBS,or a peroxidase-labeled anti-human IgG antibody (manufactured byAmerican Qualex) which had been diluted 3,000 times, was dispensed in 50μl portions into the wells to carry out the reaction at room temperaturefor 1 hour, the resulting plate was washed with 0.05% Tween-PBS, andthen an ABTS [ammonium 2,2-azinobis(3-ethylbenzothiazole-6-sulfonate)]substrate solution was dispensed in 50 μl portions into the wells todevelop color and measure its absorbance at OD_(415 nm) using Emax(manufactured by Molecular Devices).

The results are shown in FIG. 33. The anti-human VEGF receptor Flt-1mouse monoclonal antibodies KM1732 and KM1750 and anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 bound toFlt-1 7N, Flt-1 3N and Flt-1 2N, but not to Flt-1 7N.K2 and KDR 7N. Inconsequence, it was confirmed that the epitope which is recognized byanti-human VEGF receptor Flt-1 mouse monoclonal antibodies KM1732 andKM1750 and anti-human VEGF receptor Flt-1 human chimeric antibodiesKM2532 and KM2550 is contained in a region of from 100 to 204 positionsof human Flt-1 counting from the N-terminal amino acid including asignal sequence.

Next, the binding activity was examined using an immunoassay method byfixing the amount of each of Flt-1 7N, Flt-1 3N, Flt-1 2N, Flt-1 7N.K2and KDR 7N to be adsorbed on a 96 well plate for EIA and varying theconcentration of human chimeric antibody to be added. Each of Flt-1 7N,Flt-1 3N, Flt-1 2N, Flt-1 7N.K2 and KDR 7N diluted to 4 μg/ml with PBSwas dispensed in 50 μl portions into wells of a 96 well plate for EIA(manufactured by Greiner) and allowed to stand overnight at 4° C. forits adsorption. After washing the plate with PBS, 100 μl of 1% BSA-PBSwas added to each well, and the remaining active groups was blocked bycarrying out the reaction at room temperature for 1 hour. Afterdiscarding 1% BSA-PBS, each of the purified anti-human VEGF receptorFlt-1 human chimeric antibodies KM2532 and KM2550 having a concentrationof from 0.0152 to 100 ng/ml was dispensed in 50 μl portions into theresulting wells to carry out the reaction at room temperature for 1hour. After washing the plate with 0.05% Tween-PBS, a peroxidase-labeledanti-human IgG antibody (manufactured by American Qualex) which had beendiluted 3,000 times with 1% BSA-PBS was dispensed in 50 μl portions intothe wells to carry out the reaction at room temperature for 1 hour, theresulting plate was washed with 0.05% Tween-PBS, and then an ABTS[ammonium 2,2-azinobis(3-ethylbenzothiazole-6-sulfonate)] substratesolution was dispensed in 50 μl portions into the wells to develop colorand measure its absorbance at OD_(415 nm) using Emax (manufactured byMolecular Devices).

The results are shown in FIG. 34 (upper drawings). The anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 bound toFlt-1 7N, Flt-1 3N and Flt-1 2N in an antibody concentration dependentfashion, but not to Flt-1 7N.K2 and KDR 7N. In addition, the anti-humanVEGF receptor Flt-1 human chimeric antibodies KM2532 and KM2550 showedalmost the same binding activity.

Next, the binding activity of human chimeric antibodies was examinedusing an immunoassay method by varying the amount of each of Flt-1 7N,Flt-1 3N, Flt-1 2N, Flt-1 7N.K2 and KDR 7N to be adsorbed on a 96 wellplate for EIA. Each of the soluble human VEGF receptors Flt-1 7N, Flt-13N, Flt-1 2N, Flt-1 7N.K2 and KDR 7N diluted to a concentration of from0.041 to 10 μg/ml with PBS was dispensed in 50 μl portions into wells ofa 96 well plate for EIA (manufactured by Greiner) and allowed to standovernight at 4° C. for its adsorption. After washing the plate with PBS,100 μl of 1% BSA-PBS was added to each well, and the remaining activegroups was blocked by carrying out the reaction at room temperature for1 hour. After discarding 1% BSA-PBS, each of the purified anti-humanVEGF receptor Flt-1 human chimeric antibodies KM2532 and KM2550 having aconcentration of 5 μg/ml was dispensed in 50 μl portions into theresulting wells to carry out the reaction at room temperature for 1hour. After washing of the plate with 0.05% Tween-PBS, aperoxidase-labeled anti-human IgG antibody (manufactured by AmericanQualex) which had been diluted 3,000 times with 1% BSA-PBS was dispensedin 50 μl portions into the wells to carry out the reaction at roomtemperature for 1 hour, the resulting plate was washed with 0.05%Tween-PBS, and then an ABTS [ammonium2,2-azinobis(3-ethylbenzothiazole-6-sulfonate)] substrate solution wasdispensed in 50 μl portions into the wells to develop color and measureits absorbance at OD_(415 nm) using Emax (manufactured by MolecularDevices).

The results are shown in FIG. 34 (lower drawings). The anti-human VEGFreceptor Flt-1 human chimeric antibodies KM2532 and KM2550 showedconcentration dependent binding activity upon the human VEGF receptorsFlt-1 7N, Flt-1 3N and Flt-1 2N, but did not react with Flt-1 7N.K2 and.KDR 7N. In addition, the anti-human VEGF receptor Flt-1 human chimericantibodies KM2532 and KM2550 showed almost the same binding activity.

3. Preparation of Anti-human VEGF Receptor Flt-1 Human CDR GraftedAntibody

An anti-human VEGF receptor Flt-1 human CDR-grafted antibody havingactivities similar to the biological activities of anti-human VEGFreceptor Flt-1 mouse monoclonal antibodies KM1732 and KM1750 andanti-human VEGF receptor Flt-1 human chimeric antibody KM2532 and KM2550was prepared.

(1) Construction of cDNA Encoding VH of Anti-human VEGF Receptor Flt-1Human CDR-grafted Antibody Based on Common Sequence of VH of Known HumanAntibodies

According to Kabat et al. (Sequences of Proteins of ImmunologicalInterest), known human antibody VHs are classifiable into subgroups I toIII [Human Sub Groups (HSG) I to III] based on the homology of aminoacid sequences, and common sequences [Human Most Homologous ConsensusSequence (hereinafter referred to as “HMHCS”)] have been identified forrespective subgroups. An amino acid sequence of VH of an anti-human VEGFreceptor Flt-1 human CDR-grafted antibody was designed based on HMHCS.For selecting HMHCS to serve as the base, the homology was examinedbetween the amino acid sequence of FR of HMHCS of human antibody VH ofeach subgroup and amino acid sequences of FR of VH of anti-human VEGFreceptor Flt-1 mouse monoclonal antibodies KM1732 and KM1750 (Table 3).

TABLE 3 Homology (%) between amino acid sequences of FR of HMHCS ofhuman antibody VH of each subgroup and amino acid sequences of FR of VHof KM1732 and KM1750 HSG I HSG II HSG III KM1732 65.5 51.7 55.2 KM175067.8 49.4 54.0

As a result, it was confirmed that KM1732 and KM1750 shows the highesthomology with subgroup I. Thus, based on the HMHCS of subgroup I, ananti-human VEGF receptor Flt-1 human CDR-grafted antibody was designed,and the cDNA encoding the amino acid sequence was constructed by usingthe polymeraze chain reaction method (PCR) in the following manner.

The construction of cDNA encoding VH of an anti-human VEGF receptorFlt-1 human CDR-grafted antibody derived from the VH of anti-human VEGFmouse receptor Flt-1 mouse monoclonal antibody KM1732 is explainedbelow. Six synthetic DNAs having a base sequence of SEQ ID NO:29 to 34were synthesized (manufactured by Sawady Technology). The DNAssynthesized each was added, to have a final concentration of 0.1 μM, to50 μl of 1×Ex Taq buffer (manufactured by Takara Shuzo Co., Ltd.)containing 200 μM dNTP, 0.5 μM M13primer RV, 0.5 μM M13primer M4 and 2.5units of TaKaRa Ex Taq DNA polymerase (all manufactured by Takara ShuzoCo., Ltd.), the mixture was covered with 50 μl of mineral oil, DNAthermal cycler PJ480 (manufactured by Perkin Elmer) was loaded with themixture, and 30 PCR cycles (at 94° C. for 2 minutes, at 55° C. for 2minutes and at 72° C. for 2 minutes per cycle) were conducted. Thereaction mixture was purified using QIAquick PCR Purification Kit(manufactured by Qiagen) according to the manual attached hereto, andthe mixture was purified and eluted with 20 μl of sterilized water.Next, the obtained elution was added to 30 μl of a buffer solutioncomprising 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mMDTT, 20 units of a restriction enzyme ApaI (manufactured by Takara ShuzoCo., Ltd.) were further added thereto, and then the reaction was carriedout at 37° C. for 1 hour. The reaction mixture was precipitated withethanol, the precipitate was added to 10 μl of a buffer solutioncomprising 50 mM Tris-ECl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride, 1 mM DTT, 100 μg/ml BSA, and 0.01% Triton X-100, 10units of restriction enzyme NotI (manufactured by Takara Shuzo Co.,Ltd.) were further added thereto, and then the reaction was allowed toproceed at 37° C. for 1 hour. The reaction mixture was fractionated byagarose gel electrophoresis, and about 0.5 μg of an ApaI-NotI fragmentabout 0.44 kb was recovered.

To 10 μl in total volume of sterilized water, 0.1 μg of the ApI-NotIfragment derived from plasmid pBluescript SK(−) obtained in 2(1) ofExample 2, and 0.1 μg of the ApaI-NotI fragment which was the PCRamplified fragment obtained in the above were added, and the fragmentswere linked using DNA ligation Kit Ver. 2 (manufactured by Takara ShuzoCo., Ltd.) according to the manual attached hereto. Escherichia coliDH5α (manufactured by TOYOBO CO., LTD.) was transformed by using thethus-obtained recombinant plasmid DNA solution. Plasmid DNA was preparedfrom each of ten E. coli transformants. As a result that the nucleotidesequence was determined according to the method described in 1(4) ofExample 2, plasmid phKM1732HV0 shown in FIG. 26 and containing cDNAencoding the amino acid sequence of interest was obtained. Thenucleotide sequence and the amino acid sequence of the VH of theanti-human VEGF receptor Flt-1 human CDR-grafted antibody (hereinafterreferred to as “1732HV0”) contained in phKM1732HV0 are shown in SEQ IDNOS:35 and 90, respectively.

The construction of cDNA encoding VH of an anti-human VEGF receptorFlt-1 human CDR-grafted antibody derived from the VH of the anti-humanVEGF mouse receptor Flt-1 mouse monoclonal antibody KM1750 is explainedbelow. The reactions were carried out in the same manner as describedabove using six synthetic DNAs having a base sequence of SEQ ID NO:36 to41 were synthesized (manufactured by Sawady Technology) to obtainplasmid phKM1750HV0 containing cDNA encoding an amino acid sequence ofinterest shown in FIG. 13. The nucleotide sequence and the amino acidsequence of the VH of the anti-human VEGF receptor Flt-1 humanCDR-grafted antibody (hereinafter referred to as “1750HV0”) contained inphKM1750HV0 are shown in SEQ ID NOS:42 and 91, respectively.

(2) Construction of cDNA Encoding VL of Anti-human VEGF Receptor Flt-1Human CDR-grafted Antibody Based on Common Sequence of VL of Known HumanAntibodies

According to Kabat et al. (Sequences of Proteins of ImmunologicalInterest), known human antibody VLs are classifiable into subgroups I toIV (HSG I to IV) based on the homology of amino acid sequences, andHMHCS has been identified for respective subgroups. An amino acidsequence of VL of an anti-human VEGF receptor Flt-1 human CDR-graftedantibody was designed based on HMHCS. For selecting HMHCS to serve asthe base, the homology was examined between the amino acid sequence ofFR of HMHCS of human antibody VL of each subgroup and amino acidsequences of FR of VL of anti-human VEGF receptor Flt-1 mouse monoclonalantibodies KM1732 and KM1750 (Table 4).

TABLE 4 Homology (%) between amino acid sequences of FR of HMHCS ofhuman antibody VH of each subgroup and the amino acid sequences of FR ofVH of KM1732 and KM1750 HSG I HSG II HSG III HSGIV KM1732 66.2 57.5 62.563.8 KM1750 68.8 63.8 68.8 70.0

As a result, it was confirmed that KM1732 shows the highest homologywith subgroup I and KM1750 shows almost the same high homology withsubgroups I and IV, so that the amino acid sequence of the VL ofanti-human VEGF receptor Flt-1 human CDR-grafted antibody was designedbased on the HMHCS of subgroup I in the case of KM1732 and the HMHCS ofsubgroups I and IV in the case of KM1750, and cDNA encoding the aminoacid sequence was constructed by PCR in the following manner.

The construction of cDNA encoding VL of an anti-human VEGF receptorFlt-1 human CDR-grafted antibody derived from the VL of anti-human VEGFmouse receptor Flt-1 mouse monoclonal antibody KM1732 is explainedbelow. Six synthetic DNAs having a base sequence of SEQ ID NO:43 to 48were synthesized (manufactured by Sawady Technology). The DNAssynthesized each was added, to have a final concentration of 0.1 μM, to50 μl of 1×Ex Taq buffer (manufactured by Takara Shuzo Co., Ltd.)containing 200 μM dNTP, 0.5 μM M13primer RV, 0.5 μM M13primer M4 and 2.5units of TaKaRa Ex Taq DNA polymerase (all manufactured by Takara ShuzoCo., Ltd.), the mixture was covered with 50 μl of mineral oil, DNAthermal cycler PJ480 (manufactured by Perkin Elmer) was loaded with themixture, and 30 PCR cycles (at 94° C. for 2 minutes, at 55° C. for 2minutes and at 72° C. for 2 minutes per cycle) were conducted. Thereaction mixture was purified using QIAquick PCR Purification Kit(manufactured by Qiagen) according to the manual attached hereto, andthe mixture was purified and eluted with 20 μl of sterilized water.Next, the obtained elution was added to 30 μl of a buffer solutioncomprising 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 1 mM DTT,and 100 μg/ml BSA, 10 units of a restriction enzyme EcoRI (manufacturedby Takara Shuzo Co., Ltd.), 10 units of a restriction enzyme SplL(manufactured by Takara Shuzo Co., Ltd.) were further added thereto, andthen the reaction was carried out at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis, and about 0.5μg of an EcoRI-SplI fragment about 0.39 kb was recovered.

To 10 μl in total volume of sterilized water, 0.1 μg of the EcoRI-SplIfragment derived from plasmid phKM1259LV0 obtained in 2(1) of Example 2,and 0.1 μg of the EcoRI-SplI fragment which was the PCR amplifiedfragment obtained in the above were added, and the fragments were linkedusing DNA ligation Kit Ver. 2 (manufactured by Takara Shuzo Co., Ltd.)according to the manual attached hereto. Escherichia coli DH5α(manufactured by TOYOBO CO., LTD.) was transformed by using thethus-obtained recombinant plasmid DNA solution. Plasmid DNA was preparedfrom each of ten E. coli transformants. As a result that the nucleotidesequence was determined according to the method described in 1(4) ofExample 2, plasmid phKM1732LV0 shown in FIG. 28 and containing cDNAencoding the amino acid sequence of interest was obtained. Thenucleotide sequence and the amino acid sequence of the VL of theanti-human VEGF receptor Flt-1 human CDR-grafted antibody (hereinafterreferred to as “1732LV0”) contained in phKM1732LV0 are shown in SEQ IDNOS:49 and 92, respectively.

When the HMHCS of human subgroup IV was used as the base in constructingcDNA encoding the VL of anti-human VEGF receptor Flt-1 human CDR-graftedantibody derived from the VL of anti-human VEGF receptor Flt-1 mousemonoclonal antibody KM1750, plasmid phKM1750LV0 (IV) containing cDNAencoding the amino acid sequence of interest shown in FIG. 29 wasobtained by carrying out the reaction similar to the above using 6synthetic DNA fragments (manufactured by Sawady Technology) havingnucleotide sequences of SEQ ID NOS:50 to 55. Corresponding nucleotidesequence and amino acid sequence of the VL of anti-human VEGF receptorFlt-1 human CDR-grafted antibody contained in phKM1750LV0 (IV)[hereinafter referred to as “1750LV0(IV)”] are shown in SEQ ID NOS:56and 93, respectively. When the HMHCS of human subgroup I was used as thebase, plasmid phKM1750LV0 (I) containing cDNA encoding the amino acidsequence of interest shown in FIG. 36 was obtained by carrying out thereaction similar to the above using 6 synthetic DNA fragments(manufactured by Sawady Technology) having nucleotide sequences of SEQID NOS:65 to 70. Corresponding nucleotide sequence and amino acidsequence of the VL of anti-human VEGF receptor Flt-1 human CDR-graftedantibody contained in phKM1750LV0 (I) [hereinafter referred to as“1750LV0 (I)”] are shown in SEQ ID NOS:71 and 94, respectively.

(3) Construction of Anti-human VEGF Receptor Flt-1 Human CDR-graftedAntibody Expression Vectors Based on HMHCS of V Regions of Known HumanAntibodies

Expression vectors of anti-human VEGF receptor Flt-1 human CDR-graftedantibody were constructed in the following manner using tandem cassettevector, pKANTEX93, for humanized antibody expression described in WO97/10354 and plasmids phKM1732HV0, phKM1732LV0, phKM1750HV0, phKM1750LV0(I) and phKM1750LV0 (IV) containing cDNA encoding the V region ofanti-human VEGF receptor Flt-1 human CDR-grafted antibody obtained in3(1) and (2) of Example 2.

The construction of an expression vector of anti-human VEGF receptorFlt-1 human CDR-grafted antibody derived from anti-human VEGF receptorFlt-1 mouse monoclonal antibody KM1732 is explained below.

To 10 μl of a buffer solution comprising 50 mM Tris-HCl (pH 7.5), 10 mMmagnesium chloride and 1 mM DTT, 5 μg of plasmid phKM1732HV0 was added,10 units of a restriction enzyme ApaI (manufactured by Takara Shuzo Co.,Ltd.) were further added thereto, and then the reaction was carried outat 37° C. for 1 hour. The reaction mixture was precipitated withethanol, the precipitate was added to 10 μl of a buffer solutioncomprising 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride, 1 mM DTT, 100 μg/ml BSA, and 0.01% Triton X-100, 10units of a restriction enzyme NotI (Takara Shuzo Co., Ltd.) were furtheradded thereto, and then the reaction was carried out at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 0.5 μg of an ApaI-NotI fragment about 0.44 kbwas recovered.

To 10 ∥l in total volume of sterilized water, the ApaRI-NotI fragmentobtained in Example 2(1) derived from tandem cassette vector forhumanized antibody expression, pKANTEX93, and 0.1 μg of the ApaI-NotIfragment of phKM1732HV obtained in the above were added, and thefragments were linked using DNA ligation Kit Ver. 2 (manufactured byTakara Shuzo Co., Ltd.) according to the manual attached hereto.Escherichia coli DH5α (manufactured by TOYOBO CO., LTD.) was transformedby using the thus-obtained recombinant plasmid DNA solution to obtainplasmid pKANTEX1732HV0 shown in FIG. 30.

Next, 3 μg of the thus obtained plasmid pKANTEX1732HV0 was added to abuffer solution comprising 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 unitsof a restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.)and 10 units of a restriction enzyme SplI (manufactured by Takara ShuzoCo., Ltd.) were added thereto, and the reaction was carried out at 37°C. for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 1 μg of an EcoI-SplI fragment about 13.20 kbin size was recovered. Further, 5 μg of plasmid phKM1732LV0 was added toa buffer solution comprising 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, 10 unitsof a restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.)and 10 units of a restriction enzyme SplI (manufactured by Takara ShuzoCo., Ltd.) were added thereto, and the reaction was carried out at 37°C. for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 0.5 μg of an EcoI-SplI fragment about 0.39 kbin size was recovered.

To 10 μl in total volume of sterilized water, 0.1 μg of the thusobtained EcoRI-SplI fragment derived from plasmid pKANTEX1732HV0 and 0.1μg of the EcoI-SplI fragment derived from plasmid pKM1732LV0 were added,and the fragments were ligated using DNA ligation Kit Ver. 2(manufactured by Takara Shuzo Co., Ltd.) according to the manualattached hereto. Escherichia coli DH5α (manufactured by TOYOBO CO.,LTD.) was transformed by using the thus-obtained recombinant plasmid DNAsolution to obtain an expression vector of anti-human VEGF receptorFlt-1 human CDR-grafted antibody, pKANTEX1732HV0LV0, shown in FIG. 31.

Regarding the construction of expression vectors of anti-human VEGFreceptor Flt-1 human CDR-grafted antibody originated from anti-humanVEGF receptor Flt-1 mouse monoclonal antibody KM1750, the reactionsimilar to the above was carried out to obtain vectors for anti-humanVEGF receptor Flt-1 human CDR-grafted antibody expression,pKANTEX1750HV0LV0 (IV) and pKANTEX1750HV0LV0 (I), shown in FIGS. 32 and37, respectively.

4. Analysis of Amino Acid Sequence of FR of V Region of Anti-human VEGFReceptor Flt-1 Human CDR-grafted Antibody and Construction of ExpressionVector for Modified Human CDR-grafted Antibody in which Amino Acids ofFR are Modified

(1) Analysis of Amino Acid Sequence of FR of V Region of Anti-human VEGFReceptor Flt-1 Human CDR-grafted Antibody

Among amino acid residues which are different between the amino acidsequence of the FR of the V region of each anti-human VEGF receptorFlt-1 human CDR-grafted antibody and the amino acid sequence of the FRof the V region of each anti-human VEGF receptor Flt-1 mouse monoclonalantibody, the positions of the amino acid residues which were consideredto be important for binding to human VEGF receptor Flt-1 in the aminoacid sequence of the FR of the V region of each anti-human VEGF receptorFlt-1 human CDR-grafted antibody were identified by constructing andcomparing a computer three-dimensional structure model of each of the Vregion of anti-human VEGF receptor Flt-1 mouse monoclonal antibodyKM1732 and KM1750 and that of each of the anti-human VEGF receptor Flt-1human CDR-grafted antibodies prepared in 3 of Example 2. As a result,regarding the anti-human VEGF receptor Flt-1 human CDR-grafted antibodyderived from anti-human VEGF receptor Flt-1 mouse monoclonal antibodyKM1732, it was considered that the positions of 24-position alanine,27-position tyrosine, 40-position alanine, 67-position arginine,69-position threonine, 70-position isoleucine, 82-position glutamic acidand 93-position valine in the amino acid sequence of the VH of SEQ IDNO:90 and the positions of 39-position proline, 45-position leucine,46-position leucine, 69-position asparatic acid, and 70-positionphenylalanine in the amino acid sequence of VL of SEQ ID NO:92 aretaking an important role in its binding to the human VEGF receptorFlt-1.

Regarding the anti-human VEGF receptor Flt-1 human CDR-grafted antibodyderived from anti-human VEGF receptor Flt-1 mouse monoclonal antibodyKM1750, it was considered that the positions of 3-position glutamine,67-position arginine, 82-position glutamic acid, 84-position serine and95-position tyrosine in the amino acid sequence of the VH of SEQ IDNO:91 and the positions of 17-position aspartic acid, 18-positionarginine, 39-position proline, 59-position serine, 69-position asparticacid and 70-position phenylalanine in the amino acid sequence of the VLof SEQ ID NO:93 are taking an important role in its binding to the humanVEGF receptor Flt-1. These facts suggest that the activity of anti-humanVEGF receptor Flt-1 human CDR-grafted antibody to bind to human VEGFreceptor Flt-1 can be modified, for example, by modifying the justdescribed positions of amino acid residues of anti-human VEGF receptorFlt-1 human CDR-grafted antibody into the amino acid residues of thecorresponding positions of anti-human VEGF receptor Flt-1 mousemonoclonal antibody.

(2) Construction of cDNA Encoding VH of Modified Human CDR-graftedAntibody in which Amino Acids of FR are Modified

Regarding the H chain of anti-human VEGF receptor Flt-1 humanCDR-grafted antibody derived from anti-human VEGF receptor Flt-1 mousemonoclonal antibody KM1750, the 3 residues (82-position glutamic acid,84-position serine and 95-position tyrosine) among the above-described 5amino acid residues considered to be taking an important role in itsbinding to the human VEGF receptor Flt-1 were considered to beparticularly important, so that cDNA encoding an amino acid sequencewhich was converted into the corresponding residues (82-positionglutamine, 84-position arginine and 95-position phenylalanine) found inKM1750 was constructed by PCR in the following manner.

Using 6 synthetic DNA fragments having the nucleotide sequences of SEQID NOS:72 to 77 (manufactured by Sawady Technology), an MroI-ApaIfragment of about 0.39 kb was recovered by PCR in accordance with theabove-described method.

Plasmid phKM1750HV0 was treated with restriction enzymes ApaI and MroIto recover an ApaI-MroI fragment of about 3.04 kb which was subsequentlyconnected with the PCR amplified fragment obtained in the above, andthen Escherichia coli DH5α (manufactured by TOYOBO CO., LTD.) wastransformed using the thus obtained recombinant plasmid DNA solution.Plasmid was prepared from each of ten E. coli transformants and itsnucleotide sequence was determined in accordance with the methoddescribed in 1(4) of Example 2 to obtain plasmid phKM1750HV3 shown inFIG. 38 which contains cDNA encoding the amino acid sequence ofinterest.

Corresponding nucleotide sequence and amino acid sequence of the VH ofanti-human VEGF receptor Flt-1 human CDR-grafted antibody contained inphKM1750HV3 (hereinafter referred to as “1750HV3”) are shown in SEQ IDNOS:78 and 95, respectively.

(3) Construction of cDNA Encoding VL of Modified Human CDR-graftedAntibody in which Amino Acids of FR are Modified

Regarding L chain of anti-human VEGF receptor Flt-1 human CDR-graftedantibody derived from anti-human VEGF receptor Flt-1 mouse monoclonalantibody KM1750, the 4 residues (17-position aspartic acid, 18-positionarginine, 69-position aspartic acid and 70-position phenylalanine) amongthe above-described 6 amino acid residues considered to be taking animportant role in its binding to the human VEGF receptor Flt-1 wereconsidered to be particularly important in KM1750LV0 (I), so that cDNAencoding an amino acid sequence which was converted into thecorresponding residues (17-position glutamic acid, 18-position glutamic,69-position phenylalanine and 70-position tyrosine) found in KM1750 wasconstructed by PCR in the following manner.

Using 6 synthetic DNA fragments having the nucleotide sequences of SEQID NOS:79 to 84 (manufactured by Sawady Technology), the same reactionwas carried out to obtain the plasmid phKM1750LV4 shown in FIG. 39 whichcontains cDNA encoding the amino acid sequence of interest.

Corresponding nucleotide sequence and amino acid sequence of the VL ofanti-human VEGF receptor Flt-1 human CDR-grafted antibody contained inphKM1750LV4 (hereinafter referred to as “1750LV4”) are shown in SEQ IDNOS:85 and 96, respectively.

(4) Construction of Expression Vector for Modified Human CDR-graftedAntibody in which Amino Acids of FR are Modified

Using plasmids phKM1750HV3 and phKM1750LV4 obtained in 4(3) of Example 2containing cDNA encoding the V region of anti-human VEGF receptor Flt-1human CDR-grafted antibody and plasmids phKM1750HV0, phKM1750LV0 (I) andphKM1750LV0 (IV) obtained in 3(1) and (2) of Example 2, the reactionsimilar to that in 3(3) of Example 2 was carried out to obtainpKANTEX1750HV3LV0 (I), pKANTEX1750HV3LV0 (IV), pKANTEX1750HV0LV4, andpKANTEX1750HV3LV4 as expression vectors of modified anti-human VEGFreceptor Flt-1 human CDR-grafted antibodies in which amino acids of FRwere modified shown in FIGS. 40, 41, 42, and 43, respectively.

Amino acid sequences of KM1750 mouse H chain, KM1750HV0 and KM1750HV3are shown in FIG. 48. Amino acid sequences of KM1750 mouse L chain,KM1750LV0 (I), KM1750LV0 (IV) and KM1750LV4 are shown in FIG. 49.

Escherichia coli DH5α/pHKM1750HV0 which harbors recombinant plasmidphKM1750HV0, Escherichia coli DH5α/phKM1750HV3 which harbors recombinantplasmid phKM1750HV3, Escherichia coli XL1-Blue/phKM1750LV0 (I) whichharbors recombinant plasmid phKM1750LV0 (I), Escherichia coliXL1-Blue/phKM1750LV0 (IV) which harbors recombinant plasmid phKM1750LV0(IV), and Escherichia coli DH5α/phKM1750LV4 which harbors recombinantplasmid phKM1750LV4 have been deposited on May 12, 1999, in NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology, and have been assigned the designations as FERMBP-6719, FERM BP-6720, FERM BP-6716, FERM BP-6717, and FERM BP-6718,respectively.

5. Production and Activity Evaluation of Anti-human VEGF Receptor Flt-1Human CDR-grafted Antibody

(1) Expression of Anti-human VEGF Receptor Flt-1 Human CDR-graftedAntibody in Rat Myeloma YB2/0 Cells (ATCC CRL1581) UsingpKANTEX1750HV0LV0 (I), pKANTEX1750HV0LV0 (IV), pKANTEX1750HV3LV0 (I),pKANTEX1750HV3LV0 (IV), pKANTEX1750HV0LV4 and pKANTEX1750HV3LV4

Introduction of anti-human VEGF receptor Flt-1 human CDR-graftedantibody expression vectors pKANTEX1750HV0LV0 (I), pKANTEX1750HV0LV0(IV), pKANTEX1750HV3LV0 (I), pKANTEX1750HV3LV0 (IV), pKANTEX1750HV0LV4and pKANTEX1750HV3LV4 into YB2/0 cells was carried out by theelectroporation method [Cytotechnology, 3: 133 (1990)] according to 1(1)of Example 2.

A 5 μg portion of each of pKANTEX1750HV0LV0 (I), pKANTEX1750HV0LV0 (IV),pKANTEX1750HV3LV0 (I), pKANTEX1750HV3LV0 (IV), pKANTEX1750HV0LV4 andpKANTEX1750HV3LV4 obtained in 4(3) of Example 2 was introduced into4×10⁶ of YB2/0 cells, and the resulting cells were suspended in 40 ml ofRPMI1640-FCS(10) medium [RPMI1640 medium (manufactured by NissuiPharmaceutical) containing 10% fetal calf serum (FCS)] and dispensed in200 μl portions into wells of a 96 well microtiter plate (manufacturedby Sumilon). After 24 hours of culturing at 37° C. in a 5% CO₂incubator, Geneticin (hereinafter referred to as “G 418”; manufacturedby Gibco) was added to a concentration of 0.5 mg/ml, and the culturingwas continued for 1 to 2 weeks. Culture supernatants were recovered fromwells in which transformant colonies having G 418 resistance were formedand became confluent, and the activity of anti-human VEGF receptor Flt-1human CDR-grafted antibody in each culture supernatant was measured bythe enzyme immunoassay shown in 2(3) of Example 2.

Transformants which showed the activity of anti-human VEGF receptorFlt-1 human CDR-grafted antibody in their culture supernatants weresubjected to cloning by carrying out limiting dilution twice to obtaintransformant cell strains capable of producing anti-human VEGF receptorFlt-1 human CDR-grafted antibody. Examples of the transformants obtainedby introducing the expression vectors pKANTEX1750HV0LV0 (I),pKANTEX1750HV0LV0 (IV), pKANTEX1750HV3LV0 (I), pKANTEX1750HV3LV0 (IV),pKANTEX1750HV0LV4 and pKANTEX1750HV3LV4, namely transformants capable ofproducing the anti-human VEGF receptor Flt-1 human CDR-grafted antibodyoriginated from anti-human VEGF receptor Fit-1 monoclonal antibodyKM1750, include KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555,respectively, and the anti-human VEGF receptor Flt-1 human CDR-graftedantibodies produced by these transformants were named KM8550, KM8551,KM8552, KM8553, KM8554 and KM8555, respectively. Each of the thusobtained transformant cell clones showed an anti-human VEGF receptorFlt-1 human CDR-grafted antibody productivity of approximately from 0.1to 1 μg/ml.

(2) Purification of Anti-Human VEGF Receptor Flt-1 Human CDR-GraftedAntibody From Culture Supernatant

Each of the anti-human VEGF receptor Flt-1 human CDR-graftedantibody-producing cell lines KM8550, KM8551, KM8552, KM8553, KM8554 andKM8555 obtained in 5(1) of Example 2 was suspended in GIT medium(manufactured by Nippon Pharmaceutical) containing 0.5 mg/ml of G 418,to a cell density of 1 to 2×10⁵ cells/ml, and the suspension wasdispensed in 200 ml portions into a total of five 175 cm² flasks(manufactured by Greiner). When the cells became confluent after 5 to 7days of culturing at 37° C. in a 5% CO₂ incubator, about 1.1 to 2.5 L ofeach culture supernatant was recovered. A column was packed with about 1ml of ProSep A (manufactured by Bioprocessing) and washed with 10 ml of1 M glycine-0.15 M NaCl (pH 8.6) at a flow rate of 1 ml/minute. Afterwashing, 2.3 L, 2.5 L, 1.9 L, 2.4 L, 1.1 L or 2 L of the culturefiltrate of anti-human VEGF receptor Flt-1 human CDR-graftedantibody-producing cell line KM8550, KM8551, KM8552, KM8553, KM8554 orKM8555, respectively, prepared in the above was passed through theProSep A column at a flow rate of 70 ml/hour. After washing with 10 mlof 1 M glycine-0.15 M NaCl (pH 8.6) at a flow rate of 1 ml/minute, thecolumn was further washed step by step with 4 ml each of 50 mM citratebuffer of pH 6, 5 and 4 in that order and then 7 ml of 50 mM citratebuffer (pH 3.0) was passed through the column to elute the humanCDR-grafted antibody. As the results, anti-human VEGF receptor Flt-1human CDR-grafted antibodies KM8550, KM8551, KM8552, KM8553, KM8554 andKM8555 were obtained in amounts of 1.1 mg, 1.8 mg, 1.6 mg, 2.2 mg, 1.3mg and 1.6 mg, respectively.

The thus purified anti-human VEGF receptor Flt-1 human CDR-graftedantibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555 wereanalyzed by the SDS-PAGE method shown in 2(3) of Example 2. Each of theanti-human VEGF receptor Flt-1 human CDR-grafted antibodies KM8550,KM8551, KM8552, KM8553, KM8554 and KM8555, the anti-human VEGF receptorFlt-1 human chimeric antibody KM2550 shown in 2(4) of Example 2 andKM8969 (Japanese Published Unexamined Patent Application No. 257893/98)as a control human CDR-grafted antibody was applied to theelectrophoresis in an amount of 2 μg protein per lane under reducing andnon-reducing conditions, and then the gels were stained with CoomassieBrilliant Blue. The results are shown in FIG. 44. The human CDR-graftedantibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555 showed aband of IgG at a position of about 150 kDa under the non-reducingcondition, and a band of H chain at a position of about 50 kDa and aband of L chain at about 25 kDa under the reducing condition. Thisresult coincided with that the IgG type antibody is separated into two Hchains and L chains under the reducing conditions caused by the cuttingof intermolecular disulfide bond and present as a molecule of 150 kDa.

(3) Binding Activities of Anti-Human VEGF Receptor Flt-1 HumanCDR-Grafted Antibodies Upon Human VEGF Receptor Flt-1

The activity of purified anti-human VEGF receptor Flt-1 humanCDR-grafted antibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555and anti-human VEGF receptor Flt-1 human chimeric antibody KM2550 tobind to the human VEGF receptor Flt-1 was confirmed according to themethod in 2(5) of Example 2.

Amount of Flt-1 7N to be coated on the plate was fixed to 2 μg/ml, andconcentration of each of the purified antibodies was varied from 1.23 to900 ng/ml (3-fold serial dilutions).

The results are shown in FIG. 45. The anti-human VEGF receptor Flt-1human CDR-grafted antibodies KM8550, KM8551, KM8552, KM8553, KM8554 andKM8555 and anti-human VEGF receptor Flt-1 human chimeric antibody KM2550bound to the human VEGF receptor Flt-1 7N in an antibody concentrationdependent fashion. In addition, the anti-human VEGF receptor Flt-1 humanCDR-grafted antibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555and anti-human VEGF receptor Flt-1 human chimeric antibody KM2550 showedalmost the same binding activity. In consequence, it was revealed thatthe six human CDR-grafted antibodies of the present invention keep thebinding activity of human chimeric antibody KM2550.

Next, in order to examine binding specificity of anti-human VEGFreceptor Flt-1 human CDR-grafted antibodies KM8550, KM8551, KM8552,KM8553, KM8554 and KM8555 and anti-human VEGF receptor Flt-1 humanchimeric antibody KM2550, their reactivity for five soluble human VEGFreceptor derivative proteins Flt-1 7N, Flt-1 3N, Flt-1 2N (WO 98/22616),Flt-1 7N.K2 [2(6-5) of Example 2] and KDR 7N [2(6-5) of Example 2] wasexamined using an enzyme immunoassay. Concentration of each solublehuman VEGF receptor derivative protein to be adsorbed on a 96 well platefor EIA was fixed to 10 μg/ml (3-fold dilution), and the antibodyconcentration was fixed to 5 μg/ml.

The results are shown in FIG. 46. The anti-human VEGF receptor Flt-1human CDR-grafted antibodies KM8550, KM8551, KM8552, KM8553, KM8554 andKM8555 and anti-human VEGF receptor Flt-1 human chimeric antibody KM2550showed almost the same reaction specificity, reacting with Flt-1 7N,Flt-1 3N and Flt-1 2N and not reacting with Flt-1 7N.K2 and KDR 7N. Theanti-GM₂ human CDR-grafted antibody KM8969 (Japanese PublishedUnexamined Patent Application No. 257893/98) used as a negative controlantibody did not react with the soluble human VEGF receptor derivativeproteins tested. In consequence, it was revealed that the six humanCDR-grafted antibodies of the present invention keep the bindingspecificity of human chimeric antibody KM2550.

Next, the activity of anti-human VEGF receptor Flt-1 human CDR-graftedantibodies KM8550, KM8551, KM8552, KM8553, KM8554 and KM8555 andanti-human VEGF receptor Flt-1 human chimeric antibody KM2550 to inhibitbinding between human VEGF and human VEGF receptor Flt-1 was examined inaccordance with the method described in 2(5) of Example 2. Concentrationof each antibody to be added was varied from 0.0048 to 15 μg/ml (5-folddilutions). The results are shown in FIG. 47. As shown in FIG. 47,anti-human VEGF receptor Flt-1 human CDR-grafted antibodies KM8550,KM8551, KM8552, KM8553, KM8554 and KM8555 and anti-human VEGF receptorFlt-1 human chimeric antibody KM2550 inhibited the binding between humanVEGF and human VEGF receptor Flt-1 in an antibody concentrationdependent fashion. In consequence, it was revealed that the six humanCDR-grafted antibodies of the present invention also keep the VEGF-Flt-1binding inhibition activity of human chimeric antibody KM2550.

(4) VEGF-Dependent Cell-Migration Inhibition Test Using Anti-VEGFReceptor Flt-1 Monoclonal Antibody

Effect of anti-VEGF receptor Flt-1 monoclonal antibody on theVEGF-dependent migration activity of human vascular endothelial cells,as an index of in vitro angiogenesis activity, was examined.

The cell migration test was carried out in accordance with the method ofSato et al. [J. Cell Biology, 107: 1199 (1988)]. HUVEC cultured in a 3.5cm dish until they became confluent was scratched with a razor's edgeand then washed with PBS. A 1.5 ml portion of a 5% FCS-containing M-199medium was added thereto, and VEGF (final concentration: 10 ng/ml) andthe anti-VEGF receptor Flt-1 monoclonal antibody KM1750 or KM1732obtained in 5 of Example 1 (final concentration: 0, 1 or 10 μg/ml) werefurther added thereto, followed by culturing for 24 hours. Afterculturing, the number of cells migrated from the scratched position wasmeasured.

As a result, cell migration ability of HUVEC increased by adding VEGF,but the migration was completely inhibited by adding the anti-VEGFreceptor Flt-1 monoclonal antibody KM1750 or KM1732 (finalconcentration: 1 μg/ml). Thus, it was confirmed that Flt-1 is a mainreceptor relating to the migration of vascular endothelial cells.

FIG. 50 shows results of the comparison of activities of anti-VEGFreceptor Flt-1 monoclonal antibodies KM1750 and KM1732 to inhibit themigration of vascular endothelial cells. Both of the monoclonalantibodies showed the activity to inhibit the migration of vascularendothelial cells concentration-dependently at a monoclonal antibodyconcentration of 0.1 to 1 μg/ml.

Accordingly, it was found that migration of vascular endothelial cellsinduced by VEGF is completely inhibited by the anti-VEGF receptor Flt-1monoclonal antibody.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese application No. Hei 8-311109 filedon Nov. 21, 1996, International application No. PCT/JP97/04259 filed onNov. 21, 1997, Japanese application No. Hei. 10-139000 filed on May 20,1998, and International application No. PCT/JP99/02661 filed on May 20,1999, the entire contents of which are incorporated hereinto byreference.

1. A method of treating a disease in which the morbid state progressesby abnormal angiogenesis, comprising administering to a human or ananimal in need of such treatment an effective amount of a recombinantmonoclonal antibody selected from the group consisting of a humanizedantibody and an antibody fragment thereof, said recombinant monoclonalantibody specifically binding to the human VEGF receptor Flt-1, andwherein the recombinant monoclonal antibody binds to an epitope presentin a region of the 100^(th) to 204^(th) positions from the N-terminalamino acid including a signal sequence of the human VEGF receptor Flt-1,and said recombinant monoclonal antibody comprises an antibody heavychain (H chain) variable region (V region), and an antibody light chain(L chain) variable region (V region), and wherein said H chain V regioncomprises the amino acid sequence of SEQ ID NO:91.
 2. A method oftreating a disease in which the morbid state progresses by abnormalangiogenesis, comprising administering to a human or an animal in needof such treatment an effective amount of a recombinant monoclonalantibody selected from the group consisting of a humanized antibody andan antibody fragment thereof, said recombinant monoclonal antibodyspecifically binding to the human VEGF receptor Flt-1, and wherein therecombinant monoclonal antibody binds to an epitope present in a regionof the 100^(th) to 204^(th) positions from the N-terminal amino acidincluding a signal sequence of the human VEGF receptor Flt-1, and saidhumanized antibody is selected from the group consisting of a humanchimeric antibody and a human complementarity-determining region(CDR)-grafted antibody, and wherein the CDR-grafted antibody comprises:CDR in a heavy chain (H chain) variable region (V region) comprising theamino acid sequences of SEQ ID NOs:15, 16, and 17, and CDR in a lightchain (L chain) variable region (V region) comprising the amino acidsequences of SEQ ID NOs:18, 19 and
 20. 3. The method according to claim2, wherein the human CDR-grafted antibody comprises: the H chain Vregion comprising the amino acid sequence of SEQ ID NO:91, and the Lchain V region comprising the amino acid sequence of SEQ ID NO:93, 94 or96.
 4. The method according to either claim 1 or 2, wherein the antibodyfragment is selected from the group consisting of Fab, Fab′, F(ab′)₂, asingle chain antibody, and a disulfide stabilized antibody.